CN104067944B - Rice fertility regulates and controls construct and its transformation event and application - Google Patents

Abstract

The present invention relates to molecular biology of plants and breeding field.Specifically, embodiment of the present invention is related to the transgenic rice plant of the plant genome DNA containing seeding technique event and positioned at transgenic sequence flank.More particularly it relates to the fertility restorer and application thereof of homozygous recessive Genetic male-sterile line plant.Further the present invention relates to building male sterible series of rice, keep the method and transformation event of system, more specifically, the present invention relates to a kind of constructs, a kind of rice cell, tissue or organ, a method of building male sterible series of rice, a method of restoring rice sterile plant male fertile, a method of preparing rice paddy seed, a kind of transformation event, a kind of rice conversion event 7R-949D, a kind of rice conversion event 7R-1425D, it is a kind of for detecting the primer of rice conversion event, it is a kind of for detecting the kit of rice conversion event, specifically, the method identified the bond area of the T-DNA and plant genome DNA that are inserted into using the kit and further identify the seed and other tissues of transformation event, a method of being used to prepare hybrid rice, and male sterility of rice Tie up to the purposes in preparation hybrid rice.

Description

Rice fertility regulates and controls construct and its transformation event and application

Technical field

The present invention relates to molecular biology of plants and breeding field.Specifically, embodiment of the present invention is related to containing system The transgenic rice plant of kind technology event and the plant genome DNA positioned at transgenic sequence flank.More specifically, of the invention It is related to the fertility restorer and application thereof of homozygous recessive Genetic male-sterile line plant.Further the present invention relates to building rice males Sterile line, the method and transformation event for keeping system, more particularly it relates to a kind of construct, a kind of rice cell, group It knits or organ, a method of building male sterible series of rice, a method of restoring rice sterile plant male fertile, it is a kind of The method for preparing rice paddy seed, a kind of transformation event, a kind of rice conversion event 7R-949D, a kind of rice conversion event 7R- 1425D, it is a kind of for detecting the primer of rice conversion event, it is a kind of for detecting the kit of rice conversion event, specifically, The bond area of the T-DNA and plant genome DNA that are inserted into using kit identification and the kind for further identifying transformation event The method of sub and other tissues, a method of hybrid rice and male sterible series of rice, which are used to prepare, in preparation hybridizes water Purposes in rice.

Background technique

The most commonly used is " three systems " and " two systems " hybridization in paddy rice cross breeding breeding." three systems " hybridization needs specific restorer It is that the procedure of breeding and production link are complicated with holding, period length, the low efficiency of breeding new sterile line and Combination nova, germ plasm resource Utilization rate be lower than 5%.In addition, triple crossing rice hybrid vigor is weaker, sterile cytoplasm is more single, and there are certain to destroy venereal disease The potential danger that insect pest is broken out." two systems " hybrid rice is due to not by restorer, keeping relationship between system to be restricted, the something lost of parent Pass diversity be improved significantly, select High-Yielding Hybrid Rice combination speed obviously accelerate, promote grinding for super hybridized rice Study carefully and produces.But the sterile line used in current " two systems " hybridization is mostly " light is temperature sensitive " sterile line, and fertility is by the temperature in environment Degree and illumination effect.The unstable purity and quantity that will have a direct impact on hybrid seed of these environmental factors increases risk in hybrid seed production, Enterprise and peasant can be made to cause heavy economic losses when serious, limitation " two systems " hybrid paddy rice is widely applied.And utilize mesh The two-line hybrid rice sterile line that preceding technology can be selected is extremely limited, such as almost without good double-line hybrid in japonica rice variety Combination, limits making full use of for variety source.Thus, cultivate it is not affected by environment and can autonomous replication stable sterile line As the widely applied technical bottleneck of limitation " two systems " hybridization technique.

Thus, current paddy rice cross breeding technology still has much room for improvement.

Expression of the foreign gene in plant is influenced by its insertion point in the plant genome, this is likely to be Due to the chromatin Structure (structure of such as heterochromatin) or neighbouring transcription regulatory element (such as enhancer) around insertion point Adjusting caused by (Weising et al., Foreign genes in plants:transfer, structure, Expression, and applications. (1988) Ann.Rev.Genet22:421-477), for example, it can be observed that Between the different numerous strains of the insertion point that gene transformation obtains, there are biggish differences for the expression of foreign gene Not, can also be observed that foreign gene, there are the differential expressions of space-time in different transformation plants, and this species diversity is not artificial Caused by the expression cassette of the expression regulation elements such as the promoter of selection building.Meanwhile foreign gene is in the difference of Plant Genome The integration of position can impact the whole phenotype of plant, for example, the insertion of foreign gene in the plant genome can make to insert The expression of plant endogenous genes at angle of striking receives influence.Therefore, during the initiative of transformation event, it is necessary to be produced into Thousand independent transformation strains up to ten thousand, by screening a large amount of transformation plant, identification obtain meeting industrialized requirement one is optimal Change event has the exogenous origin gene integrator site to meet the requirements and expression/mode, while not making to other phenotypes of plant At influence.Further, it is possible to which the foreign gene of the optimization event is passed through hybridization by the conventional breeding methods of backcross transformation Transformation is into the kind of other genetic backgrounds, and the transgene expression that the filial generation of these hybrids has been assigned initial transformant is special Property, meanwhile, also maintain the various merits of original kind.

In the filial generation of plant or seed or its filial generation or sexual hybridization, the presence of the specific transformation event of specific detection or There is no being very important, event-specific detection method can be identified between the exogenous DNA of insertion and acceptor gene group solely Special engagement (junction), this is directed not only to transgenosis itself, further relates to it in the genome of host plant or seed Position is integrated in insertion.In addition, for detect the method for particular event for permit before the listing of vegetable food product and the regulation of label, Or the character etc. of crop is also very helpful in environmental monitoring and monitoring field.

Summary of the invention

The present invention is directed to solve one of above-mentioned technical problem at least to a certain extent or at least provide one kind useful quotient Industry selection.For this purpose, an object of the present invention is to provide a kind of with the recessive rice hero that can effectively construct novel stabilising Property sterile line and make full use of Rice Germplasm Resources for crossbreeding, improve purity of hybrid means.

The present invention is the following discovery based on inventor and completes: inventor is prominent with homozygous recessive Genetic male-sterile line Variant is transformation receptor material, and 3 target genes of close linkage are converted into the male sterile rice mutant receptor plant.Institute Stating 3 target genes is rice fertility restorer genes, pollen inactivated gene and color mark screening-gene respectively.Wherein, fertility Restoring gene can make the transformation receptor fertility restorer of infertility, and pollen inactivated gene can be such that the pollen of the foreign gene containing conversion loses It is living, that is, fertilizing ability is lost, screening-gene can be used for the sorting of transgenic seed and non-transgenic seed, non-turn sorted out Gene seed continuously, steadily to produce sterile as sterile line production cenospecies, transgenic seed as holding system System.For example, according to one embodiment of present invention, it can be using the recessive infertility ms26/ms26 mutant of rice core as transformation receptor Material converts 3 target genes of close linkage to the sterile line: where restoring gene OsCYP704B2(is corresponding wild The rice MS26 gene of raw type) transformation receptor fertility restorer can be made；Pollen inactivated gene Zm-AA1 can make containing foreign gene Pollen inactivation, that is, lose fertilizing ability；Point of the fluorescence color sorting gene DsRed (r) for transgenic seed and non-transgenic seed It picks, the non-transgenic seed sorted out is used as sterile line and produces cenospecies, and transgenic seed, which is used as, keeps system to come continuously Steadily produce sterile line.Since the technology produces non-transgenic product using biotechnology, solves the paddy rice cross breeding production of hybrid seeds The bottleneck problem faced in journey, i.e. three line method resource utilization are low and in two line method the problem of sterile line fertility instability.

As a result, in one embodiment of the present of invention, the invention proposes a kind of constructs.According to an embodiment of the invention, The construct includes: the first expression cassette, and first expression cassette contains the first nucleic acid molecules, the first nucleic acid molecule encoding water Rice fertility restorer gene；And second expression cassette, second expression cassette contain the second nucleic acid molecules, second nucleic acid Molecule encoding pollen inactivated gene.Using the construct, effectively male sterility of rice restoring gene and pollen can be inactivated Gene is introduced into homozygous recessive Genetic male-sterile line mutant plants, to obtain the fertile plant conduct of foreign gene-carrying System is kept, is so as to continuously produce sterile line conveniently by selfing and keep, in addition, not foreign gene-carrying Plant may be used as hybridization female parent.Thus, it is possible to be efficiently used for paddy rice cross breeding, obtained cenospecies is also non-to turn base Cause.

Thus, it is possible to by routine techniques, such as agrobacterium-mediated transformation, by preceding construct be introduced into rice cell, In tissue or organ, to obtain that the sample of research, hybridization can be subsequently used for.Thus, in the second aspect of the present invention, this hair It is bright to propose a kind of rice cell, tissue or organ.According to an embodiment of the invention, containing in the rice cell, tissue or organ There is mentioned-above construct.

In the third aspect of the present invention, the invention proposes a kind of methods for constructing male sterible series of rice.According to this hair Bright embodiment, this method comprises: mentioned-above construct is introduced into the first rice homozygous recessive male sterile plants, To obtain the second rice plant of foreign gene-carrying, second rice plant can generate fertile males gamete, therefore It is able to carry out self-fertilization, the seed of the seed of available foreign gene-carrying and not foreign gene-carrying, the two respectively accounts for 50%.Wherein, the seed of foreign gene-carrying not can be used as male sterible series of rice.It is thus possible to conveniently by selfing Continuously production sterile line and holding system, in addition, the plant of foreign gene-carrying not may be used as the parent of hybridization.By This, can be efficiently used for paddy rice cross breeding.

In the fourth aspect of the present invention, the invention proposes a kind of methods for restoring rice sterile plant male fertile.Root According to the embodiment of the present invention, this method comprises: mentioned-above construct is introduced into rice homozygous recessive male sterile plants In.

In the fifth aspect of the invention, the invention proposes a kind of methods for preparing rice paddy seed.Reality according to the present invention Example is applied, method includes the following steps: mentioned-above construct is introduced into rice plant；And by the rice plant Self-fertilization, to obtain the seed for containing mentioned-above construct.In the sixth aspect of the present invention, the invention proposes one kind Transformation event.According to an embodiment of the invention, the transformation event is pure by the way that mentioned-above construct is introduced into rice It is obtained in the recessive male sterile plants of conjunction, wherein the construct includes: the first expression cassette, and first expression cassette contains First nucleic acid molecules, the first nucleic acid molecule encoding male sterility of rice restoring gene；And second expression cassette, described second Expression cassette contains the second nucleic acid molecules, the second nucleic acid molecule encoding pollen inactivated gene.It, can be effective using the construct Male sterility of rice restoring gene and pollen inactivated gene are introduced into homozygous recessive Genetic male-sterile line mutant plants by ground In, so that the fertile plant for obtaining foreign gene-carrying is as holding, so as to continuously give birth to conveniently by selfing It produces sterile line and keeps system, in addition, the plant of foreign gene-carrying not may be used as the female parent of hybridization.Thus, it is possible to effectively For paddy rice cross breeding.Thus, it is possible to by routine techniques, such as agrobacterium-mediated transformation, preceding construct is introduced into rice In cell, tissue or organ, to obtain that the sample of research, hybridization can be subsequently used for.

In the seventh aspect of the present invention, the invention proposes a kind of rice conversion event 7R-949D.Reality according to the present invention Example is applied, at least one selected from SEQ ID NO:13,14,17,18 and 53 is included in the genome of rice conversion event 7R-949D Kind DNA sequence dna.As a result, according to an embodiment of the invention, the invention proposes a kind of plants, wherein the plant includes rice Transformation event 7R-949D.It is contained in the genome of the plant selected from SEQ ID NO:13,14,17,18 and 53 at least A kind of DNA sequence dna or its complementary series.And the invention proposes the seed obtained by the plant derivation, cell and tissues.

In the eighth aspect of the present invention, the invention proposes a kind of rice conversion event 7R-1425D.It is according to the present invention Embodiment, in the genome of rice conversion event 7R-1425D comprising selected from SEQ ID NO:15,16,19,20 and 54 extremely A kind of few DNA sequence dna.As a result, according to an embodiment of the invention, the invention proposes a kind of plants, wherein the plant includes Rice conversion event 7R-1425D.It is contained in the genome of the plant selected from SEQ ID NO:15,16,19,20 and 54 At least one DNA sequence dna or its complementary series.And the invention proposes the seed obtained by the plant derivation, cell and groups It knits.

In the ninth aspect of the present invention, the invention proposes a kind of for detecting the primer of rice conversion event.According to this The embodiment of invention, for detecting the primer of rice conversion event 7R-949D, which is characterized in that the primer includes to be selected from SEQ At least one of ID NO:13,14,17,18,53 or its complementary series.For detecting drawing for rice conversion event 7R-1425D Object, which is characterized in that the primer includes at least one selected from SEQID NO:15,16,19,20,54 or its complementary series.

In the tenth aspect of the present invention, the invention proposes a kind of for detecting the kit of rice conversion event.According to The embodiment of the present invention, the kit include mentioned-above primer.

Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description Obviously, or practice through the invention is recognized.

Detailed description of the invention

Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures Obviously and it is readily appreciated that, in which:

Fig. 1 is the structural schematic diagram according to the plant expression vector p7R of one embodiment of the invention.Wherein, from right margin, Successively contain PG47 promoter:: ZM-BT1 leads peptide:: ZM-AA1 gene:: IN2-1 terminator expression cassette, OsCYP704B2 base Because of expression cassette and END2 promoter:: DsRed (r) gene:: PINII terminator expression cassette.

Fig. 2 shows the I2-IK of fertile flower powder according to an embodiment of the invention and abortive pollen grain (not fertile) Coloration result, wherein 7R-949D indicates the coloration result of 7R-949D；The coloration result of 7R-1425D expression 7R-1425D.

Fig. 3 is T-DNA insertion point and Integration Mode schematic diagram in transformation event 7R-949D.In the transformation event In genome, 2 T-DNA are incorporated, the T-DNA1 and T-DNA2 respectively illustrated.

Fig. 4 is T-DNA insetion sequence and contiguous gene positional relationship and verifying primer signal in transformation event 7R-949D Figure, wherein SP3 is the primer according to T-DNA sequence design, and A949B-L-1 and A949B-R are the base according to insertion point flank Because of the primer of group sequence design, it is 981bp that wherein primer A949B-L-1 and SP3, which carries out the clip size of PCR amplification acquisition, Specific nucleotide sequence is as shown in SEQ ID NO:74；The clip size of primer A949B-R and SP3 progress PCR amplification acquisition For 540bp, specific nucleotide sequence is as shown in SEQ ID NO:75.

Fig. 5 is T-DNA insertion point and inserted mode schematic diagram in transformation event 7R-1425D.In the transformation event In genome, 1 T-DNA, right margin RB, left margin LB are incorporated.

Fig. 6 is T-DNA insetion sequence and contiguous gene positional relationship and verifying primer signal in transformation event 7R-1425D Figure.Wherein 7RB-3 and SP3 is the primer according to T-DNA sequence design, and A1425RB-2 and A1425LB-2 are according to insertion point The primer of the genome sequence design of flank, wherein primer A1425RB-2 and SP3 carry out the clip size of PCR amplification acquisition and are 864bp, specific nucleotide sequence is as shown in SEQ ID NO:76；A1425LB-2 and SP3 carries out the piece of PCR amplification acquisition Duan great little is 954bp, and specific nucleotide sequence is as shown in SEQ ID NO:77.

Fig. 7 is that probe location and restriction enzyme site schematic diagram, 7A show purpose base on T-DNA in transformation event 7R-949D Because of upper probe sequence position and Hind III digestion site；7B shows I digestion of probe sequence position and EcoR on color sorting gene Site.

Fig. 8 is transformation event 7R-1425D using OsCYP704B2 as the expection hybridized fragment size of probe and Hind III enzyme Enzyme site schematic diagram.

It is the expected hybridized fragment size of probe and EcoR I that Fig. 9, which is transformation event 7R-1425D with Zm-AA1 and DsRed (r), Restriction enzyme site schematic diagram.

Figure 10 be transformation event 7R-949D T2, T3 and T4 for plant using target gene as the Southern blot of probe As a result, wherein track 1,7 and 13: molecular weight standard；2,8 and 14: positive control (Plasmid DNA)+force transports No. 7 DNA-of round-grained rice BamHI；3,9 and 15: negative control (force fortune No. 7 ms26/ms26 mutant DNA-BamHI of round-grained rice)；4,5 and 6:7R-949D-Hind III-T2 3 independent transgenosis single plants of generation；10,11 and 12:7R-949D-Hind III-T3,3 independent transgenosis lists of generation Strain；16,17 and 18:7R-949D-Hind III-T4,3 independent transgenosis single plants of generation.

Figure 11 be transformation event 7R-949D T2, T3 and T4 for plant using color sorting gene as the Southern blot of probe As a result, wherein track 1,7 and 13: molecular weight standard；2,8 and 14: positive control (Plasmid DNA)+force transports No. 7 DNA-of round-grained rice BamHI；3,9 and 15: negative control (force fortune No. 7 ms26/ms26 mutant DNA-BamHI of round-grained rice)；4,5 and 6:7R-949D-EcoR I-T2 3 independent transgenosis single plants of generation；10,11 and 12:7R-949D-EcoR I-T3,3 independent transgenosis single plants of generation； 16,17 and 18:7R-949D-EcoR I-T4,3 independent transgenosis single plants of generation.

Figure 12 be transformation event 7R-1425D T2, T3 and T4 for plant using OsCYP704B2 gene as probe Southern blot is as a result, wherein track 1,7 and 13: molecular weight standard；2,8 and 14: positive control (Plasmid DNA)+force Transport No. 7 DNA-BamHI of round-grained rice；3,9 and 15: negative control (force fortune No. 7 ms26/ms26 mutant DNA-BamHI of round-grained rice)；4,5 and 6: 7R-1425D-Hind III-T2 3 independent transgenosis single plants of generation；10,11 and 12:7R-1425D-Hind III-T3 generation 3 Independent transgenosis single plant；16,17 and 18:7R-1425D-Hind III-T4,3 independent transgenosis single plants of generation.

Figure 13 be transformation event 7R-1425D T2, T3 and T4 for plant using Zm-AA1 gene as the Southern of probe Blot is as a result, wherein track 1,7 and 13: molecular weight standard；2,8 and 14: positive control (Plasmid DNA)+force transports round-grained rice 7 DNA–BamHI；3,9 and 15: negative control (force fortune No. 7 ms26/ms26 mutant DNA-BamHI of round-grained rice)；4,5 and 6:7R- 1425D-EcoR I-T2 3 independent transgenosis single plants of generation；10, in 11 and 12:7R-1425D-EcoR I-T3 generation 3, is independent Transgenosis single plant；16,17 and 18:7R-1425D-EcoR I-T4,3 independent transgenosis single plants of generation.

Figure 14 be transformation event 7R-1425D T2, T3 and T4 for plant using DsRed (r) gene as the Southern of probe Blot is as a result, wherein track 1,7 and 13: molecular weight standard；2,8 and 14: positive control (Plasmid DNA)+force transports round-grained rice 7 DNA–BamHI；3,9 and 15: negative control (force fortune No. 7 ms26/ms26 mutant DNA-BamHI of round-grained rice)；4,5 and 6:7R- 1425D-EcoR I-T2 3 independent transgenosis single plants of generation；10, in 11 and 12:7R-1425D-EcoR I-T3 generation 3, is independent Transgenosis single plant；16,17 and 18:7R-1425D-EcoR I-T4,3 independent transgenosis single plants of generation.

Figure 15 is RT-PCR identification T2 for 7R-949D3 target gene expression pattern figure of transformation event, and wherein root is seedling stage Root；Stem is seedling stage stem；Leaf is seedling stage leaf；The P3 phase is clever flower primordium idiophase children's fringe；When the P6 phase is pollen mother cells Phase children's fringe；The P8 phase is pollen maturation phase children's fringe；Seed is the seed of kernel maturity stage；Mutant is military fortune No. 7 ms26/ of round-grained rice ms26；Wild type is military fortune round-grained rice 7；It is amplification template that blank control, which is with water,；Positive control is to be with transformant genomic DNA Expand template.

Figure 16 is RT-PCR identification T2 for 7R-1425D3 target gene expression pattern figure of transformation event, and wherein root is seedling Phase root；Stem is seedling stage stem；Leaf is seedling stage leaf；The P3 phase is clever flower primordium idiophase children's fringe；The P6 phase is pollen mother cells Period children's fringe；The P8 phase is pollen maturation phase children's fringe；Seed is the seed of kernel maturity stage；Mutant is military fortune No. 7 ms26/ of round-grained rice ms26；Wild type is military fortune round-grained rice 7；It is amplification template that blank control, which is with water,；Positive control is to be with transformant genomic DNA Expand template.

Figure 17 shows according to an embodiment of the present invention, the recessive infertility ms26/ms26 mutant of rice core be convert by Body material obtains the schematic diagram of sterile line by selfing by the obtained transformant of transgenosis, and wherein receptor (ms/ms) refers to pure Close recessive nucleus male sterility transgenic acceptor；System is kept to contain homozygous recessive kernel male sterile site and transgenosis heterozygosis position Point, therefore be fertile；Sterile line contains homozygous recessive kernel male sterile site and without transgenosis, therefore is male sterility；It protects Holding is that the pollen half of generation contains transgenosis, and half does not contain transgenosis；Keep tying in fact generate 50% male-sterile seed and 50% maintainer seed.

Detailed description of the Invention

The embodiment of the present invention is described below in detail.The embodiments described below with reference to the accompanying drawings are exemplary, purport It is being used to explain the present invention, and is being not considered as limiting the invention.

All bibliography being mentioned herein all are incorporated herein by reference.

Unless there are indicating on the contrary, all technical and scientific terms used herein all have common with fields of the present invention The identical meaning that technical staff is generally understood.Unless there are indicating on the contrary, technology that is used herein or mentioning is ability Standard technique well known to the those of ordinary skill of domain.Material, method and example are only used as to illustrate, rather than limit.

Term " event " refers to that original transformant and the transformant including allogeneic dna sequence DNA include but is not limited to pass through selfing or miscellaneous The filial generation that friendship or vegetative propagation generate.By with allogeneic dna sequence DNA, that is, the nucleic acid construct including intended transgenic is to plant cell It is converted, the regeneration of the plant population generated in specified plant genome, and selection is inserted by transgenosis to specific base Because of the specific plant that the insertion of group position is characterized, to generate transgenosis " event ".Thus, term " event " also refer to by Transformant and another include that sexual cutcross is carried out between heterologous transgene DNA and the kind of flanking genomic dna is raw The filial generation of production.Term " event " also refers to DNA's from original transformant, comprising insertion the and DNA for being closely adjacent to insertion The DNA of flanking genomic sequence, waits in expectation and is transferred into filial generation, a parent of the filial generation as the DNA for including insertion It is that (for example, filial generation of original transformant and selfing or vegetative propagation) and the parental department progress of the DNA without containing the insertion are sexual Hybridization as a result, receiving the insertion DNA including intended transgenic.

Term " first ", " second " be used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance or Implicitly indicate the quantity of indicated technical characteristic." first " is defined as a result, the feature of " second " can be expressed or imply Ground includes one or more of the features.In the description of the present invention, the meaning of " plurality " is two or more, unless Separately there is clearly specific restriction.

The present invention is the following discovery based on inventor and completes: inventor is to turn with rice core recessiveness sterile mutant Change acceptor material, by converting 3 target genes of close linkage into sterile mutant, wherein restoring gene can Make transformation receptor fertility restorer, pollen inactivated gene can be such that the pollen containing foreign gene inactivates, that is, lose fertilizing ability, screening Gene can be used for the sorting of transgenic seed and non-transgenic seed, and the non-transgenic seed sorted out is produced as sterile line Cenospecies, transgenic seed be used as keep system come continuously, steadily produce sterile line.For example, according to the present invention one A embodiment, can be using the recessive infertility ms26/ms26 mutant of rice core as transformation receptor material, by 3 mesh of close linkage Genetic transformation is marked to sterile line: restoring gene OsCYP704B2 can make transformation receptor fertility restorer, pollen inactivated gene Zm- AA1 can be such that the pollen containing foreign gene inactivates, that is, lose fertilizing ability, and fluorescence color sorting gene DsRed (r) is used for transgenosis kind The sorting of son and non-transgenic seed, the non-transgenic seed sorted out are used as sterile line and produce cenospecies, and transgenic seed is used Make keep system come continuously, steadily produce sterile line.Since the technology produces non-transgenic product using biotechnology, Solves the bottleneck problem faced during the paddy rice cross breeding production of hybrid seeds, i.e., three line method resource utilization is low and sterile line is educated in two line method The unstable problem of property.

As a result, in one embodiment of the present of invention, the invention proposes a kind of constructs.According to an embodiment of the invention, The construct includes: the first expression cassette, and first expression cassette contains the first nucleic acid molecules, the first nucleic acid molecule encoding water Rice fertility restorer gene；And second expression cassette, second expression cassette contain the second nucleic acid molecules, second nucleic acid Molecule encoding pollen inactivated gene.Using the construct, effectively male sterility of rice restoring gene and pollen can be inactivated Gene is introduced into rice plant such as rice homozygous recessive male sterile plants, to obtain the fertile plant of foreign gene-carrying As system is kept, so as to continuously produce sterile line conveniently by selfing and keep system.In addition, not carrying external source The plant of gene may be used as the parent among hybridization.Thus, it is possible to be efficiently used for paddy rice cross breeding.

Herein, the form of construct is not particularly limited, specific example according to the present invention, can for plasmid, Bacteriophage, artificial chromosome, clay (Cosmid), viral at least one.Specific example according to the present invention, construct (have When also referred to as expression vector, genetic carrier or carrier) in the form of plasmid.Plasmid as genetic carrier, have it is easy to operate, can To carry the property of larger segment, convenient for operating and handling.The form of plasmid is also not particularly limited, either annular matter Grain, be also possible to linear plasmid, it can be it is single-stranded, be also possible to double-strand.Those skilled in the art can according to need It is selected.According to an embodiment of the invention, Ti carrier can be used, such as can be arranged using by the first and second expression cassettes Between the right boundary of the T-DNA of expression vector p7R.Thus, it is possible to pass through Agrobacterium-medialed transformation method for the first He Second expression cassette is converted into recipient plant, such as rice ms26 recessive nucleus male sterility mutant.Thus, it is possible to be free of The rice conversion strain of herbicide resistance markers' gene and antibiotic-resistance marker's gene.The transformation plant so obtained has as follows Feature: (1) it converts site and is in heterozygous state always in each generation, therefore have half pollen without foreign gene, half contains Foreign gene, the pollen containing foreign gene inactivates (losing fertilizing ability), so foreign gene is only transferred to by oogamete The next generation, will not be by pollen dispersal into environment；(2) transformant selfing can be solid, ties fertile seed (band fluorescent marker) Ratio with sterile seed (without fluorescent marker) is 1:1, and fertile plant (having foreign gene), which is used as, keeps system, can be by certainly It hands over and easily, continuously produces sterile line and keep system, sterile plant (being free of transgene component) is used as hybridization in production The parent of the production of hybrid seeds；(3) because sterile plant is free of transgenosis, transgenosis is free of with the hybrid seed of its production, is hybridized with this The rice commodity grain of kind production less contains transgenosis, to eliminate the hidden danger of GMO bio-safety.The novel hybridization is educated Kind of system is makes full use of rice heterosis to provide practicable technology new breakthrough.

Term " nucleic acid " as used in the present invention can be any comprising deoxyribonucleotide or ribonucleotide The polymer of acid, including but not limited to by modification or unmodified DNA, RNA, length is not by any special limit System.It is more stable because DNA is for RNA for the carrier for constructing recombinant cell, preferred nucleic acid DNA, and And it is easily operated.

According to an embodiment of the invention, the type of male sterility of rice restoring gene is not particularly restricted.In the present invention One embodiment in, male sterility of rice restoring gene coding has the amino acid sequence as shown in SEQ ID NO:6 Protein.I.e., it is possible to the male sterility of rice restoring gene used is OsCYP704B2, thus, it is possible to as rice by The wild type restoring gene of body ms26 Mutants homozygous (holandry infertility).The albumen category of OsCYP704B2 gene coding In cytochrome P-450 family, the specifically expressing in the suede adhesion coating in P8 to the P10 stage of anther development and microspore.The gene It will lead to the expansion of suede adhesion coating after mutation, exposore is incomplete and develops termination and the termination development of anther cuticula, so as to cause plant Strain male sterility, and female fertility is normal.Further chemical composition analysis discovery, in the anther of the deletion mutant body In, it is nearly no detectable cutin monomer, and then find that the function of the gene is that catalysis generates the hydroxyl containing 16 and 18 carbochains Fatty acid.

According to a particular embodiment of the invention, in one embodiment of the invention, the male sterility of rice restores base Because having the nucleotide sequence as shown in SEQ ID NO:5.(its nucleotide sequence is such as with the OsCYP704B2 gene of wild type Shown in SEQ ID NO:22) it compares, nucleotide sequence shown in SEQ ID NO:5 introduces three single nucleotide mutations, but not Change the amino acid sequence of its coding, position of these three single nucleotide mutations on the gene coding region OsCYP704B2 and specific Mutation is respectively as follows: 238 nucleotide A and sports C；240 nucleotide G sport C；243 nucleotide G sport C.Hair Bright people using nucleotide sequence shown in the SEQ ID NO:5 it has surprisingly been found that can be convenient for distinguishing in various Molecular Identifications Foreign gene and endogenous gene, and the fertility of rice ms26/ms26 infertility recipient plant can more effectively be made to be restored. Rice receptor ms26 Mutants homozygous is by radiation-induced gained, and mutation is (big comprising OsCYP704B2 by 3103bp missing Partial Fragment) lead to (deleted segment physical location: ensembl plants oryza japonica group Version64.6 (MSU6) chromosome3:3,701,319-3,704,421).Large fragment deletion mutation makes back mutation Probability is extremely low, therefore sterile character is stablized, to ensure the stability of sterile line, reduces hybrid seeding risk.

In one embodiment of the invention, first expression cassette can further include: the first promoter, described First promoter and first nucleic acid molecules are operably associated, and first promoter is andro gamete specificity promoter； And first terminator, first terminator and first nucleic acid molecules are operably associated.Implementation according to the present invention The type of example, the first promoter and the first terminator is not particularly restricted.According to one embodiment of present invention, for OsCYP704B2 gene can use the endogenesis promoter of OsCYP704B2, the sequence in the area ORF and terminator, be wild water Rice genome sequence.In one embodiment of the invention, first promoter has the nucleosides as shown in SEQ ID NO:7 Acid sequence.In one embodiment of the invention, first terminator has the nucleotides sequence as shown in SEQ ID NO:8 Column.It is surprisingly found by the inventors that can further improve the corresponding egg of expression significantly using the combination of the promoter and terminator White efficiency, and then can be improved the efficiency using construct building sterile line, and can more effectively make rice ms26/ The fertility of ms26 infertility recipient plant is restored.

According to an embodiment of the invention, the type of pollen inactivated gene is not particularly restricted.Implementation according to the present invention Example, the pollen inactivated gene coding have the protein of the amino acid sequence as shown in SEQ ID NO:21.Thus, it is possible to encode The encoded alpha-amylase of Zm-AA1.Alpha-amylase belongs to glycosyl hydrolase.The gene be from pollination 10 days after maize and Isolated in the cDNA library of endosperm, function is catalyzing hydrolysis polysaccharide molecule (such as starch) (1-4)-alpha-D-glucose Glycosides.The endogenous Zm-AA1 gene of corn is mainly expressed in the scultellum tissue of the seed of sprouting, can't detect this in zasiokaurin The expression of gene.According to an embodiment of the invention, the pollen inactivated gene has the nucleotides sequence as shown in SEQ ID NO:9 Column.Thus, it is possible to further increase the efficiency for expressing corresponding albumen.According to an embodiment of the invention, the second expression cassette is further It include: the second promoter, second promoter and second nucleic acid molecules are operably associated, and second promoter is Pollen specific promoter；And second terminator, second terminator and second nucleic acid molecules are operably associated. Thus, it is possible to more effectively improve the expression efficiency of corresponding gene.In addition, according to an embodiment of the invention, in the second expression cassette In can further include coding and lead the sequence of peptide, the second expression cassette can be encoded effectively with the pollen for leading peptide as a result, Inactivating protein navigate in specific organelle thus, it is possible to be targeted target gene (pollen inactivated gene) can.Example Such as, according to an embodiment of the invention, coding lead peptide sequence have the nucleotide sequence as shown in SEQ ID NO:36 (from The coding of the brittle-1 gene of corn leads the sequence of peptide (TP)).Thus, it is possible to which effectively expressed targeting proteins are formed sediment Powder decomposes the starch in pollen, so that pollen be made to lose vigor, loses fertilizing ability, transgenic pollen is caused to inactivate.Into And according to a particular embodiment of the invention, the gene is under zasiokaurin specificity promoter PG47 driving, and from corn Brittle-1 gene coding lead peptide (TP) sequence and terminator IN2-1 composition expression cassette, can be in the maturation of development later stage Specific expressed amylase in pollen, and amyloplaste is targeted, the starch in pollen is decomposed, so that pollen be made to lose vigor, is lost Fertilizing ability causes transgenic pollen to inactivate.The design cannot inseminate so that all transgenic pollen inactivations containing this gene The bio-safeties problem such as genetic drift can also be strictly prevented, the pollen of inactivation cannot pollinate with other plant around or weeds, because And transgenosis cannot be by pollen dispersal into environment.

In addition, according to an embodiment of the invention, construct can further include: third expression cassette, the third table It include third nucleic acid molecules up to box, the third nucleic acid molecule encoding screening-gene, the screening-gene is bioluminescence gene.By This, convenient for by screening-gene expression come determine plant and its part whether the gene introduced containing construct.

According to an embodiment of the invention, can be using selected from red fluorescent gene, cyan fluorescent protein gene, yellow fluorescence Protein gene, luciferase gene, green fluorescence protein gene, anthocyanin p1 gene and glufosinate transacetylase encoding gene At least one as screening-gene.It in one embodiment of the invention, can be using red fluorescent protein as screening base Cause.Red fluorescent protein gene (DsRed) derives from reef coral (Discosoma sp.), is that unique non-grain is made in expression cassette The gene order in object source.Red fluorescent protein maximum absorption wave a length of 558nm, maximum emission wavelength 583nm.By DsRed The amino acid sequence of coding is by showing that similitude is extremely low with anaphylactogen and toxalbumin sequence alignment, non-toxic and sensitization. DsRed is commonly used for the screening-gene of genetic transformation, and GMO bio-safety thing topic never occurred.In an implementation of the invention In example, the screening-gene has the nucleotide sequence as described in SEQ ID NO:1.Thus, it is possible to more efficiently express red Color fluorescin, the expression of enhancing DsRed gene in rice.Nucleotide sequence and wild type described in SEQ ID NO:1 DsRed gene (its nucleotide sequence is as shown in SEQ ID NO:23) is compared, and there are two single nucleotide mutations for tool, is named as DsRed(r).The two single nucleotide mutations, which are respectively as follows:, to be converted to G by the 21st bit base C, is converted to C by the 315th bit base G. It is surprisingly found by the inventors that red fluorescent protein can be expressed more efficiently, enhancing red fluorescent protein gene is in rice Expression.

In one embodiment of the invention, the third expression cassette further comprises: third promoter, the third open Mover and the third nucleic acid molecules are operably associated, and the third promoter is the starting of callus or seed specific Son；Third terminator, the third terminator and the third nucleic acid molecules are operably associated.In an implementation of the invention In example, the third promoter has the nucleotide sequence as shown in SEQ ID NO:2.In one embodiment of the invention, The third terminator has the nucleotide sequence as shown in SEQ ID NO:3.As a result, according to one embodiment of present invention, The opening code-reading frame of DsRed (r) is connected to from corn and is callus and seed (embryo and endosperm) specificity promoter Between END2 and terminator Pin II from potato, DsRed (r) expression casette (END2 ﹕ ﹕ DsRed (r) ﹕ ﹕ is reassembled into PINII).The red for being very easy to identification, therefore the expression cassette is presented in rice paddy seed containing the expression cassette under fluorescence excitation In the present invention for recognizing and sorting holding system and male-sterile seed.

As a result, according to an embodiment of the invention, can use the construct of embodiment according to the present invention, with non-transgenic Receptor of the recessive nucleus male sterility rice (ms26/ms26) as conversion, carries out genetic transformation, obtains integration containing following close The rice of three chain foreign gene DsRed (r), Ms26, Zm-AA1 keep system, Ms26, that is, OsCYP704B2 fertile gene. The insertion of foreign gene and endogenous male sterility site (ms26/ms26) are non-chain, therefore obtained transgenic paddy rice is protected Hold be the recessive sterile site ms26 containing independent homozygosis and heterozygosis foreign gene (including OsCYP704B2 gene) integration Site.

Thus, it is possible to by routine techniques, such as agrobacterium-mediated transformation, by preceding construct be introduced into rice cell, In tissue or organ, to obtain that the sample of research, hybridization can be subsequently used for.Thus, in the second aspect of the present invention, this hair It is bright to propose a kind of rice cell, tissue or organ.According to an embodiment of the invention, containing in the rice cell, tissue or organ There is mentioned-above construct.In one embodiment of the invention, the rice cell, tissue or organ are homozygous from rice Recessive male sterile plants.In one embodiment of the invention, the rice homozygous recessive male sterile plants include Ms26 The homozygous recessive alleles of gene.It is male can be efficiently used for building for rice cell of the invention, tissue or organ as a result, Property sterile plant.Feature and advantage described in construct are previously with regard to, are also applied for the rice cell, tissue or organ, no longer It repeats.

As a result, in the third aspect of the present invention, the invention proposes a kind of methods for constructing male sterible series of rice.According to The embodiment of the present invention, with reference to Figure 17, this method comprises: it is male that mentioned-above construct is introduced into the first rice homozygous recessive In property sterile plant, to obtain the second rice plant of foreign gene-carrying, second rice plant can generate fertile Male gamete, and the foreign gene in the second rice plant is in heterozygous state, therefore has half flower in the second rice plant Powder is free of foreign gene, and half contains foreign gene, and the pollen containing foreign gene inactivates (losing fertilizing ability).And then it cultivates Obtained second rice plant, it is available not carry external source base by the self fertilizing of the second rice plant, that is, transformant The seed of cause, to construct male sterible series of rice.According to an embodiment of the invention, the first rice homozygous recessive male sterility is planted Strain includes the homozygous recessive alleles of Ms26 gene.In addition, according to an embodiment of the invention, can be carried out by fluorescence detection The step of sorting, for example whether issuing fluorescence to be sorted, is distinguished that is, by detecting whether rice paddy seed carries bioluminescence gene Its whether foreign gene-carrying.Feature and advantage described in construct are previously with regard to, this method is also applied for, are repeated no more.

In the fourth aspect of the present invention, the invention proposes a kind of methods for restoring rice sterile plant male fertile.Root According to the embodiment of the present invention, this method comprises: mentioned-above construct is introduced into rice homozygous recessive male sterile plants In.In one embodiment of the invention, the rice homozygous recessive male sterile plants include the homozygous recessive of Ms26 gene Allele.Feature and advantage described in construct are previously with regard to, this method is also applied for, are repeated no more.

In the fifth aspect of the invention, the invention proposes a kind of methods for preparing rice paddy seed.Reality according to the present invention Example is applied, method includes the following steps: mentioned-above construct is introduced into rice plant；And by the rice plant Self-fertilization, to obtain the seed for containing mentioned-above construct.In one embodiment of the invention, the rice plant For rice homozygous recessive male sterile plants.In one embodiment of the invention, the rice homozygous recessive male sterility is planted Strain includes the homozygous recessive alleles of Ms26 gene.

In the sixth aspect of the present invention, the invention proposes a kind of transformation events.According to an embodiment of the invention, described turn Change event is obtained by the way that mentioned-above construct to be introduced into rice homozygous recessive male sterile plants.In the present invention One embodiment in, the rice homozygous recessive male sterile plants include Ms26 gene homozygous recessive alleles.? In one embodiment of the present of invention, the transformation event is at least one selected from 7R-949D and 7R-1425D.Of the invention In one embodiment, the construct is introduced by agrobacterium-mediated transformation.

According to an embodiment of the invention, the present invention utilizes Agrobacterium-mediated Transformation method by OsCYP704B2, ZM- of close linkage AA1 and DsRed (r) genetic transformation obtain 7R-949D the and 7R-1425D transgenic paddy rice strain of inheritance stability into rice System.Using TAIL-PCR technology, T1 is expanded for the flanking sequence of the insertion position plant T-DNA, obtains flanking sequence； The flanking sequence of acquisition is subjected to sequencing analysis, and with database (MSU Rice Genome Annotation Project Release7, issuing time on October 31st, 2011, ftp: //ftp.plantbiology.msu.edu/pub/data/ Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecu les/version_7.0/) in water Rice genome sequence is compared, and the T-DNA insertion point of discovery 7R-949D and 7R-1425D is respectively positioned at No. 3 dyeing (physical location Chr3:14,746,015-14,746,027) and No. 1 chromosome long arm distal end at the nearly centromere of body galianconism (physical location Chr1:42,215,016-42,215,095), is both not inserted into inside rice endogenous gene；Then, it docks It closes region and carries out PCR amplification to verify the insertion position external source T-DNA and initial guess T-DNA Integration Mode, i.e., with flanking sequence And PCR amplification is carried out for target sequence between T-DNA lateral order, as a result it is consistent with expection, further demonstrates T-DNA insertion point Correctness, and show that double copies that 7R-949D is differential concatenation are integrated, and T-DNA is single copy insertion in 7R-1425D.

As a result, in the seventh aspect of the present invention, the invention proposes a kind of rice conversion event 7R-949D.According to this hair Bright embodiment, comprising selected from SEQ ID NO:13,14,17,18 and 53 in the genome of rice conversion event 7R-949D At least one DNA sequence dna.In addition, according to an embodiment of the invention, the invention proposes a kind of plants, wherein the plant packet The 7R-949D of transformation event containing rice.It is contained in the genome of the plant selected from SEQ ID NO:13,14,17,18 and 53 At least one DNA sequence dna or its complementary series.And the invention proposes the seed obtained by the plant derivation, cell and groups It knits.In the eighth aspect of the present invention, the invention proposes a kind of rice conversion event 7R-1425D.Implementation according to the present invention , at least one selected from SEQ ID NO:15,16,19,20 and 54 is included in the genome of rice conversion event 7R-1425D Kind DNA sequence dna.In addition, according to an embodiment of the invention, the invention proposes a kind of plants, wherein the plant includes rice Transformation event 7R-1425D.It is contained in the genome of the plant selected from SEQ ID NO:15,16,19,20 and 54 extremely A kind of few DNA sequence dna or its complementary series.And the invention proposes the seed obtained by the plant derivation, cell and tissues.

In addition, coming from rice event for detecting the present invention also provides a kind of transgenic detection method and combinations thereof The plant of 7R-949D or seed, or transgenosis/genomic DNA of product of the part from the transgenic plant or seed connect Connect the detection in area.Transformation event 7R-949D, complete external source insetion sequence be as shown in SEQ ID NO:53, the area T-DNA and The catenation sequence (also known as chimeric dna molecule) that 5 ' flanking sequences of insertion point are constituted is as shown in SEQ ID NO:17, wherein the 1-10 nucleotides sequences are classified as rice endogenous gene group DNA, and 11-20 nucleotides sequences are classified as the T-DNA of external source insertion Sequence；Catenation sequence with 3 ' end side wing Sequence compositions is as shown in SEQ ID NO:18, wherein 1-10 nucleotide sequences For the T-DNA sequence of external source insertion, 11-20 nucleotides sequences are classified as rice endogenous gene group DNA.In the present invention, on Stating catenation sequence can also be on the basis of SEQ ID NO:17 and 18, including longer genomic dna sequence and external source are inserted into T-DNA sequence, more specifically, the connection sequence that 5 ' flanking sequences of external source insertion T-DNA sequence and insertion point are constituted It arranges as shown in SEQ ID NO:13, wherein the 884-903 nucleotide sequences of SEQID NO:13 such as SEQ ID NO:17 institute Show；The catenation sequence such as SEQ ID NO:14 institute that 3 ' flanking sequences of the external source insertion T-DNA sequence and insertion point are constituted Show, wherein the 497-516 nucleotide sequences of SEQ ID NO:14 are as shown in SEQ ID NO:18.All these sequences and Plants and Seeds comprising these sequences constitute one aspect of the present invention.The present invention provides a kind of new DNA sequences as a result, Column, DNA transgenosis/genome area SEQ ID NO:13, SEQ ID NO of the sequence from transformation event 7R-949D: 53, SEQ ID NO:14 or its complementary DNA molecule.It in its genome include SEQ ID NO:13, SEQ ID NO:53, SEQ The rice plant and seed of ID NO:14 or its complementary DNA molecule are within the scope of the present invention.

DNA detection the present invention also provides one group of PCR primer for transformation event 7R-949D, wherein one group of PCR Primer includes the first PCR primer and the second PCR primer, wherein the first PCR primer includes the region T-DNA of SEQ ID NO:13 Any portion of at least 11 or more continuous polynucleotides, 5 ' flank water of second PCR primer from SEQ ID NO:13 The continuous polynucleotides of any portion of similar length of rice genomic DNA region, these nucleic acid molecules are as primer molecule It is together effective when progress PCR amplification.Or first PCR primer include SEQ ID NO:14 the region T-DNA it is any Partial at least 11 or more continuous polynucleotides, 3 ' flank rice bases of second PCR primer from SEQ ID NO:14 Because of the continuous polynucleotides of any portion of similar length in group region of DNA domain, which carries out PCR expansion together It is effective when increasing.Or first PCR primer and the second PCR primer be all from SEQ ID NO:53, include sequence SEQ ID Any portion of at least 11 or more the continuous polynucleotides of NO:53, one group of PCR primer carry out PCR amplification together When be effective.The amplified production that PCR acquisition is carried out by using above-mentioned primer, can be used for detecting rice conversion event 7R- 949D.Include DNA sequence dna shown in all or part of SEQ ID NO:13,14,17,18 or 53 in the DNA cloning product.

The present invention also provides a kind of transgenic detection methods and combinations thereof, come from rice event 7R- for detecting The plant of 1425D or seed, or the part from the transgenic plant or seed product DNA transgenosis/genome connection The detection in area.Transformation event 7R-1425D, for complete external source insetion sequence as shown in SEQ IDNO:54, external source is inserted into T-DNA The catenation sequence that 5 ' flanking sequences of sequence and insertion point are constituted is as shown in SEQ ID NO:19, wherein 1-10 nucleosides Acid sequence is rice endogenous gene group DNA, and 11-20 nucleotides sequences are classified as the T-DNA sequence of external source insertion；External source insertion The catenation sequence of 3 ' end side wing Sequence compositions of T-DNA sequence and insertion point is as shown in SEQ ID NO:20, wherein 1-10 The nucleotides sequence of position is classified as the T-DNA sequence of external source insertion, and 11-20 nucleotides sequences are classified as rice endogenous gene group DNA. In the present invention, above-mentioned catenation sequence can also be on the basis of SEQ ID NO:19 and 20, including longer genomic DNA sequence The T-DNA sequence of column and external source insertion, more specifically, 5 ' flanking sequences of external source insertion the T-DNA sequence and insertion point The catenation sequence of composition can be as shown in SEQ IDNO:15, and wherein the 817-836 nucleotide sequences of SEQ ID NO:15 are such as Shown in SEQ ID NO:19；The catenation sequence that 3 ' flanking sequences of the external source insertion T-DNA sequence and insertion point are constituted is such as Shown in SEQ ID NO:16, wherein the 398-417 nucleotide sequences of SEQ ID NO:16 are as shown in SEQ IDNO:20.Institute There are these sequences and the Plants and Seeds comprising these sequences to constitute one aspect of the present invention.As a result, the present invention provides A kind of new DNA sequence dna, DNA transgenosis/genome area SEQ ID NO of the sequence from transformation event 7R-1425D: 15, SEQ ID NO:16, SEQ ID NO:54 or its complementary DNA molecule.It in its genome include SEQ ID NO:15 or SEQ The rice plant and seed of ID NO:16 or its complementary DNA molecule are within the scope of the present invention.

DNA detection the present invention also provides one group of PCR primer for transformation event 7R-1425D, this group of PCR primer packet Include third PCR primer and the 4th PCR primer.Wherein third PCR primer includes any portion in the region T-DNA of SEQ IDNO:15 At least 11 or more the continuous polynucleotides divided, 5 ' flank paddy genes of the 4th PCR primer from SEQ ID NO:15 Group region of DNA domain any portion of similar length continuous polynucleotides, these nucleic acid molecules as primer molecule together It is effective when progress PCR amplification.Or third PCR primer includes any portion of of the region T-DNA of SEQ ID NO:16 At least 11 or more continuous polynucleotides, 3 ' flank oryza sativa genomic dnas of the 4th PCR primer from SEQ ID NO:16 The continuous polynucleotides of any portion of similar length in region, this group of PCR primer carry out PCR as primer molecule together It is effective when amplification.Or third PCR primer and the 4th PCR primer are all from SEQ ID NO:54, include sequence SEQ ID Any portion of at least 11 or more the continuous polynucleotides of NO:54, this group of PCR primer carries out PCR amplification together when It is effective.The amplified production that PCR acquisition is carried out by using above-mentioned primer, can be used for detecting rice conversion event 7R- 1425D.Include DNA sequence dna shown in all or part of SEQ ID NO:15,16,19,20 or 54 in the amplified production.

Term " primer " used in herein is isolated polynucleotide, is hybridized by nucleic acid more with complementary target Nucleic acid chains anneal to form the hybrid of primer and target polynucleic acid chain, then more along target by polymerase, such as archaeal dna polymerase Nucleic acid chain extension.Primer pair of the invention be related to they for expand target polynucleic acid molecule amplification purposes, for example, passing through Polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.

Primer of the invention can hybridize with target dna sequence under strict conditions.It can be used any conventional nucleic acid miscellaneous Friendship or amplification method can be used for identifying the presence of the DNA from 7R-949D event or 7R-1425D in sample.Nucleic acid molecules or Its segment can in some cases with other nucleic acid molecules specific hybrids.As employed herein, if two nucleic acid molecules Can be formed antiparallel double-strandednucleic acid structure and has sufficient length to maintain this structure under high stringency conditions, then and referred to as two A nucleic acid molecules mutually can specifically hybridize.If nucleic acid molecules show complete complementarity, it is another for claiming nucleic acid molecules " complement " of one nucleic acid molecules.As employed herein, when each nucleotide of a molecule and the core of another molecule Thuja acid mutual added time, referred to as molecule show " complete complementary ".If the phase mutual cross of molecule have enough stability with Them are allowed to keep mutual annealing under the conditions of at least conventional " low strict ", claiming two molecules is " most low complementary ". Similarly, if the phase mutual cross of molecule has enough stability to allow them to keep under the conditions of conventional " high stringency " Mutual annealing, claiming the molecule is " complementary ".Sambrook et al.,1989,and by Haymes et al. (1985) conventional stringent condition is described.It is thus admissible from the deviation of complete complementarity, as long as this deviation is endless The ability that molecule forms duplex structure is excluded entirely.In order to make nucleic acid molecules as primer or probe, it is only necessary in the sequence sufficiently Complementation so that stable duplex structure can be formed under used specific solvent and salinity.

As employed herein, substantially homologous sequence is the nucleic acid sequence under high stringency in contrast The nucleic acid sequence of complement specific hybrid.The suitable stringent condition for promoting DNA hybridization, for example, 6.0 × sodium chloride/lemon About 45 DEG C of sour sodium (SSC), is to be washed at 50 DEG C with 2.0 × SSC later, is well known to those skilled in the art.For example, washing The salinity washed in step can be selected from low stringent about 2.0 × SSC, 50 DEG C to the stringent about 0.2 × SSC of height, 50 DEG C. In addition, the temperature in washing step can be increased to the pact of high stringency from about 22 DEG C at room temperature of Low stringency conditions 65℃.Temperature and salinity can all change or temperature or salinity remains unchanged and another variable changes.Preferred Embodiment in, nucleic acid of the invention will be specific under moderate stringency, such as at about 2.0 × SSC and about 65 DEG C The nucleic acid molecules that ground hybridization needs to expand.

About use specific amplimer to carry out target nucleic acid sequence amplification (for example, passing through PCR), " stringent item Part " is the condition for allowing primer pair only to hybridize with target nucleic acid sequence, with corresponding wild-type sequence (or its complement) Primer preferably will generate unique amplified production, amplicon in the hot amplified reaction of DNA in conjunction with the target nucleic acid sequence.

Term " being specific to (target sequence) " refer to primer under stringent hybridization conditions only with the sample comprising target sequence In target sequence hybridization.

As used herein, " DNA of amplification " or " amplicon " refer to the target nucleic acid sequence as nucleic acid-templated part The product of the nucleic acid amplification of column.For example, in order to determine whether the plant for originating from sexual hybridization contains transgenic event 7R-949D, Or whether sample of the acquisition from field includes 7R-949D or whether plant extracts includes 7R-949D.From Plant tissue samples Or the nucleic acid PCR amplification method including primer pair can be used in the DNA extracted in extract, the primer pair includes from neighbour The primer of the genome area of the insertion point for the heterologous transgene DNA being closely inserted into, and the heterologous transgene DNA from insertion The second primer, be diagnostic amplicon for the presence of event DNA to generate.Amplicon is with certain length and has sequence Column, the sequence is also diagnostic to the event.The length of amplicon can add a nucleotide base according to primer pair To or add about 50 nucleotide bases pair, or add about 250 nucleotide bases pair, or add about 350 A nucleotide base to or more pattern length and change.

Alternatively, primer pair can include whole to obtain from the flanking genomic sequence of the two sides T-DNA of insertion The amplicon of a T-DNA insertion nucleotide sequence.The member of primer pair from plant genome sequences can be selected from away from insertion Transgenosis T-DNA molecule certain distance in, which can be from a nucleotide base to about 20,000 nucleotide bases Change between pair.The use of term " amplicon " will be particularly intended to exclude the primer dimer that can be formed in the hot amplified reaction of DNA.

Any of various nucleic acid amplification reaction methods known in the art, including polymerase chain reaction can be passed through (PCR) Lai Shixian nucleic acid amplification.Various amplification methods are known in the art, other DNA expansions of these methods and this field Increasing method can be used in practice of the invention.Multiple technologies can be provided to detect the amplicon of these methods generation.One This method is Genetic Bit Ananlysis(Nikiforov, et al., 1994), wherein a DNA oligonucleotides is designed, Its DNA transgenic sequence for covering neighbouring flanking genomic DNA sequence and insertion.Oligonucleotide is fixed on microwell plate Hole in.Target area is being carried out (to use the primer and adjacent side flap genome in T-DNA insetion sequence after PCR A primer in sequence), single stranded PCR products can with fixed oligonucleotide hybridization and serve as template, for poly- using DNA The ddNTP of synthase and the label for the next base for being specific to expectation carries out single base extension.Readout can be base In fluorescence or based on ELISA signal.Signal designation as successfully expand, hybridize and Single base extension caused by insertion The presence of object/flanking genomic sequence.

Another method is Winge(2000) description Pyrosequencing technology.Design one is few in this approach Nucleotide covers neighbouring genomic DNA and insert DNA contact.Make oligonucleotides and the single-stranded PCR from target area Product (for a primer in the sequence of insertion, one in flanking genomic sequence) hybridization, there are archaeal dna polymerase, ATP, It is incubated in the case where sulfurylase, luciferase, apyrase, 5 ' phosphoric acid of adenylate and luciferin.Respectively DNTPs is added, measurement generates the incorporation of optical signal.Optical signal is indicated due to successfully expanding, hybridizing and single base or polybase The presence of transgenic insertions/flanking sequence caused by base extends.

The fluorescence polarization of Chen etc. (1999) description can be used for detecting a kind of method of amplicon of the invention.It uses This method designs an oligonucleotides, the DNA contact of covering gene group flank and insertion.Make oligonucleotides and comes from target area The single stranded PCR products (for a primer in the DNA sequence dna of insertion, one in the flanking genomic DNA sequence) in domain hybridize, and are depositing It is incubated in the case where the ddNTP of archaeal dna polymerase and fluorescent marker.Single base extension leads to the incorporation of ddNTP.Utilize fluorimeter The variation of measurement polarization can measure incorporation.The variation of polarization is indicated due to successfully expanding, hybridizing and Single base extension is led Transgenic insertions/flanking genomic sequence presence of cause.

Taqman(PE Applied Biosystems, Foster City, CA) is described as a kind of pair of DNA sequence dna In the presence of being detected and quantitative method, can be understood completely according to the explanation that producer provides.Briefly, an oligonucleotides is designed The DNA contact of probe, covering gene group flank and insertion.There are heat-stabilised poly synthase and dNTP, FRET is visited Needle and PCR primer (primer is in the DNA sequence dna of insertion and one in flanking genomic sequence) are recycled.FRET The hybridization of probe causes fluorescence part on FRET probe to crack and discharge from quencher moieties.Fluorescence signal is indicated due to successful Flanking genomes/transgenic insertions sequence presence that amplification and hybridization generate.

Such as Tyangi et al.(1996) described in, Molecular Beacons has been used for Sequence Detection In.Briefly, a FRET oligonucleotide probe is designed, flanking genomes and insert DNA contact are covered.The FRET probe Unique structure causes it to contain secondary structure, which remain adjacent to fluorescence and quencher moieties.There are thermostabilizations In the case where polymerase and dNTP, (primer is in the DNA sequence dna of insertion and one in side for FRET probe and PCR primer In wing genome sequence) it is recycled.After successful PCR amplification, FRET probe causes probe to the hybridization of target sequence The space of the elimination of secondary structure and fluorescence part and quencher moieties separates, and generates fluorescence signal.Fluorescence signal indicate due to Successfully flanking genomes/transgenic insertions sequence presence that amplification and hybridization generate.

The method of other descriptions, such as the method and apparatus that microfluidics provides separation and DNA amplification sample. Photoinitiator dye is for detecting and measuring specific DNA molecular.Comprising the electronic sensor for detecting DNA molecular or combine specific DNA molecular receive pearl and thus can be detected receive test tube (nanotube) equipment (WO/06024023) for detection the present invention DNA molecular be also useful.

As a result, in the ninth aspect of the present invention, the invention proposes a kind of for detecting the primer of rice conversion event.Root According to the embodiment of the present invention, which includes at least one selected from SEQ ID NO:13,14,15,16 and its complementary series.By This, can be reacted by PCR and effectively be detected to rice conversion event, rice conversion thing can especially be effectively detected At least one of part 7R-949D and 7R-1425D.In the tenth aspect of the present invention, the invention proposes one kind for detecting rice The kit of transformation event.According to an embodiment of the invention, the kit includes mentioned-above primer.

In the eleventh aspect of the present invention, the invention proposes a kind of methods for being used to prepare hybrid rice.According to this hair Bright embodiment, this method use male sterible series of rice, which is to construct rice male not by front The method for being is educated to construct.Thus, it is possible to further carry out paddy rice cross breeding using male sterible series of rice of the invention, water is improved The efficiency of rice hybridization.Feature and advantage described in construct are previously with regard to, this method is also applied for, are repeated no more.

In the twelveth aspect of the present invention, the invention proposes use of the male sterible series of rice in preparation hybrid rice On the way.According to an embodiment of the invention, the male sterible series of rice is the method structure for constructing male sterible series of rice by front It builds.Thus, it is possible to further carry out paddy rice cross breeding using male sterible series of rice of the invention, the effect of paddy rice cross breeding is improved Rate.Feature and advantage described in construct are previously with regard to, the purposes is also applied for, repeats no more.

In still another aspect of the invention, the invention also provides a kind of methods for constructing male sterible series of rice.According to this The embodiment of invention, this method include using rice homozygous recessive male sterile plants as maternal, the rice homozygous recessive Male sterile plants include the homozygous recessive alleles of Ms26 gene；Backcross transformation is carried out with recurrent parent by maternal, so as to Obtain the male sterible series of rice with the recurrent parent character, wherein the recurrent parent does not have the pure of Ms26 gene Close Recessive alleles.Method of the invention is utilized as a result, it can be on the basis of MS26 homozygous recessive male rice sterile line On, develop the sterile line of more different genetic backgrounds.According to an embodiment of the invention, using conventional back cross breeding method, institute Having different types of rice that can formulate into corresponding intelligent sterile line (can continuously produce corresponding sterile line Kind), so that the hybrid vigour utilization of resources is reached 95% or more.

It is described the invention also provides the composition and method of a kind of transgenic paddy rice containing specific exogenous DNA array Specific exogenous DNA array is introduced into recipient plant by the method for rice conversion, and is referred to herein as " event 7R- The event of 949D " or " 7R-949D " or " 949D " or " event 949D ".The plant of conversion or seed are referred to as " rice 7R- 949D " or " rice 949D " etc..The present invention also provides for identifying by the event 7R-949D filial generation derived or containing The material and method of the plant of the event DNA.

It is described specific the invention proposes the composition and method of the transgenic paddy rice containing specific exogenous DNA array Exogenous DNA array is introduced into recipient plant by the method for rice conversion, and is referred to herein as " event 7R-949D " Or the event of " 7R-1425D " or " 1425D " or " event 1425D ".The plant of conversion or seed are referred to as " rice 7R- 1425D " or " rice 1425D " etc..The present invention also provides for identifying by the event 7R-1425D filial generation derived or The material and method of plant containing the event DNA.

It also proposed a species specific flanking sequence in the embodiment of the present invention, " flanking sequence " is also known as " side Sequence ", in the present invention, the flanking sequence can be used for developing the spy of event 7R-949D and 7R-1425D in biological sample Anisotropic identification method.In some embodiments, the left margin of 7R-949D and 7R-1425D and the flank of right margin are also disclosed Regional sequence, these flanking sequences can be used for designing specific primer and probe.The present invention also provides be based on above-mentioned specificity Primer and probe, to the method whether identified comprising 7R-949D and 7R-1425D in biological sample.

Embodiment according to the present invention is recorded, the present invention also provides in test sample with event 7R-949D and 7R- The method that the corresponding DNA of 1425D whether there is.Specifically, which comprises (a) is by sample and DNA primer comprising DNA Contact, the DNA primer carry out core with the genomic DNA extracted from the plant comprising event 7R-949D or 7R-1425D When thuja acid amplified reaction, the specific amplicon for identifying event 7R-949D or 7R-1425D can produce；(b) nucleic acid is carried out Amplified reaction generates amplicon；(c) amplicon described in Testing and appraisal.

Contain the genome side at external source insetion sequence specific in event 7R-949D and 7R-1425D and insertion point The DNA molecular for the catenation sequence that sequence is constituted, and the sequence homologous or complementary with the DNA molecular, in guarantor of the invention Within the scope of shield.

It also proposed in embodiment of the present invention a kind of comprising flanking sequence specific in event 7R-949D or connection sequence Column, the specific DNA molecular as shown in SEQ ID NO:13,14,17 or 18, and include flank sequence specific in event 7R-1425D Column or catenation sequence, the specific DNA molecular as shown in SEQ ID NO:15,16,19 or 20.Include in embodiment of the present invention DNA sequence dna, flank rice genome of the DNA sequence dna by a transgene insert sequence and one from insertion point DNA composition, the DNA sequence dna can be used for design primer, and the primer is amplifiable to be out for detecting in plant or vegetable material The no amplicon product containing event 7R-949D or 7R-1425D.

A kind of external source insertion area T-DNA (its containing event 7R-949D is further provided in embodiment of the present invention Nucleotide sequence is as shown in SEQ ID NO:53) at least 11 or more nucleotide DNA sequence dna, and contain event 7R- At least 11 or more the nucleosides of the external source insertion area T-DNA (its nucleotide sequence is as shown in SEQ ID NO:54) of 1425D The DNA sequence dna of acid or the complementary series of above-mentioned DNA sequence dna, and shown in SEQ ID NO:13,14,17 or 18 with 7R-949D Flank rice genomic DNA sequence there is the DNA sequence dna or its complementary series of similar-length, or the SEQ with 7R-1425D Flank rice genomic DNA sequence shown in ID NO:15,16,19 or 20 has the DNA sequence dna or its complementary sequence of similar-length Column.Above-mentioned DNA sequence dna can be used as the primer sequence in DNA cloning.The amplicon as caused by above-mentioned primer can be respectively used to examine Survey event 7R-949D or 7R-1425D.Therefore, embodiment of the present invention also includes the amplicon generated by DNA primer, described DNA primer and the area transgenosis T-DNA of 7R-949D or 7R-1425D or its specific flanking sequence are homologous or complementary.

It also proposed in embodiment of the present invention in a kind of test sample corresponding to event 7R-949D or 7R-1425D The method of DNA molecular, which comprises (a) contacts the DNA sample being extracted from plants out with DNA probe, the probe Including can be with the DNA hybridization extracted from event 7R-949D or 7R-1425D but in stringency hybridization item under stringent hybridisation conditions The not molecule with adjoining tree DNA hybridization under part；(b) sample and probe is made to be in stringent hybridisation conditions；(c) detection probe with The hybridisation events of DNA.More specifically, it is additionally provided in embodiment of the present invention and corresponds to 7R-949D or 7R- in test sample The method of the specific DNA molecular of 1425D, the method includes (a) to contact probe with sample, and the sample is from rice plant In the DNA that extracts, the partial sequence of the DNA probe molecule specific sequence such as catenation sequence in event, the DNA Probe molecule under stringent hybridisation conditions can with the DNA hybridization of event 7R-949D or 7R-1425D, and in stringency hybridization item It cannot be with the DNA hybridization that compares rice plant under part；(b) sample and probe are under stringent hybridisation conditions；(c) detection probe With the hybridisation events of DNA.

Embodiment of the present invention further provides the DNA of event 7R-949D or 7R-1425D in a kind of biological sample Detection kit.The kit includes the first primer and the second primer that can be used for PCR evaluation program, and the first primer can With the left margin or right margin flanking sequence of specific recognition event 7R-949D or 7R-1425D, second primer can be special External source in anisotropic identification events 7R-949D or 7R-1425D is inserted into DNA sequence dna.Embodiment of the present invention also proposed another kind The kit of event 7R-949D or 7R-1425D in biological sample are detected, the kit includes a specific probe, described Specific probe, which has, corresponds to following sequence or the sequence complementary with following sequence: with event 7R-949D or 7R-1425D The sequence of similitude of the specific regions with 80%-100%.Probe sequence corresponding to specific regions includes event 7R- The part 5 ' of 949D or 7R-1425D or 3 ' shoulder portion sequences.

Embodiment of the present invention also proposed a kind of for other purposes and using in the embodiment of the invention addressed Method and kit, the other purposes include but is not limited to following: the event in plant identification, vegetable material or product 7R-949D or 7R-1425D, the product include but is not limited to containing or plant-derived food or feed product (it is fresh or It is finished)；In order to distinguish the transgenic line in transgenic line and non-transgenic material；And in order to determine comprising water The quality of the vegetable material of rice event 7R-949D or 7R-1425D.The kit can also be containing for examinations method Other necessary reagents and material.

On the other hand, the present invention also provides a kind of progeny plant of the production containing event 7R-949D or 7R-1425D Method.The progeny plant can be selfing or hybrid plant.In other embodiments, the invention proposes one kind to be used for thing The method of the marker-assisted breeding of part 7R-949D or 7R-1425D.On the other hand, the invention also provides one kind includes event The rice plant of the stable conversion of 7R-949D or 7R-1425D.

The polynucleotide sequence of initiative seed production technique event 7R-949D and 7R-1425D is connected in same DNA vector On, the polynucleotide sequence obtains event 7R-949D and 7R-1425D after being inserted into the specific position of rice genome.? State on chromosome location carry 7R-949D event plant contain the genome as shown in SEQ ID NO:13,14,17 or 18/ Transgene insert sequence constitute catenation sequence, on already described chromosome location carry 7R-1425D event plant contain as The catenation sequence that genome shown in SEQ ID NO:15,16,19 or 20/transgene insert sequence is constituted.With event 7R- The characteristic of the genomic insertion site of 949D or 7R-1425D is breeding efficiency to can be enhanced, and make educating using molecular labeling Transgene insert sequence is tracked in kind of groups and its filial generation to be possibly realized.The present invention also provides be used for rice event 7R-949D Or the plant of 7R-1425D, plant part, the identification of seed and cereal product, a variety of method and compositions for being detected and used.

In some embodiments, the polynucleotide sequence for formulating rice 7R-949D or 7R-1425D event can also lead to The method for crossing genetic engineering carries out molecule aggregation (molecular stack).In other embodiments, the molecule aggregate At least one other transgene polynucleotide sequences can also be further included.The polynucleotide sequence can assign the production of hybrid seeds Technology others characteristic assigns transformed plant other plant character.

In certain embodiments, polynucleotide of interest sequence can be carried out to any combination creation molecule aggregate, and Conversion plant provides the plant of required character combination to formulate, to realize the aggregation of plant trait.Heretofore described " property Shape " refers to the phenotype showed after specific dna sequence or the expression of DNA sequence dna group.The combination of the generation can also include Multiple copies of any one or more polynucleotide of interest sequences.The character aggregation combination can pass through any method Initiative including but not limited to carries out plant breeding by conventional method, or is obtained by the method for genetic transformation.If sequence It is to be assembled by Genetic Transformation in Higher Plants, then polynucleotide of interest can be combined at any time, in any direction.Institute The polynucleotide of interest sequence that stating character can be provided by conversion expression cassette introduces in cotransformation operating procedure.For example, such as Fruit will introduce two sequences, then two sequences may be embodied in different conversion expression cassettes (trans-), or be included in same In a conversion expression cassette (cis-).The expression of the sequence can by it is same or be different promoter driving.In certain feelings Condition, it may be desirable to introduce the conversion expression cassette for inhibiting the expression of polynucleotide of interest sequence.Can also by will inhibit expression cassette or It is overexpressed box and carries out any combination, to generate the plant with the combination of required character.Those skilled in the art should also be appreciated that Polynucleotide sequence can also be assembled by site-specific recombination system in required genomic locations.Above-mentioned technology ginseng See patent WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, all of the above is led to herein It crosses and is incorporated by.

It should be noted that construct according to an embodiment of the present invention and application thereof is present inventor by arduous Creative work and Optimization Work just complete.During specific embodiment is stated, unless otherwise indicated, the meaning of " plurality " is two It is a or more than two.

Specific embodiment

Below according to specific embodiment, the present invention will be described.It should be noted that these embodiments are only to be Illustrate the present invention, and limitation of the present invention cannot be construed in any way.In addition, unless stated otherwise, following Method involved in embodiment be conventional method, be referred to " Molecular Cloning:A Laboratory guide " third edition or Related product into Row.Reagents or instruments used without specified manufacturer, being can be with conventional products that are commercially available.

Embodiment 1: vector construction

By the following DNA elements of assembly, the expression vector for being referred to as p7R as shown in Figure 1 is constructed:

1) based on pCAMBIA1300 carrier, using on Xmn I and Bgl II digestion removal pCAMBIA1300 plasmid Hygromycin gene and cauliflower mosaic virus 35 S promoter sequence；

2) opening code-reading frame of expression casette END2:DsRed (r)-PINII, DsRed (r) gene (SEQ ID NO:1) It is connected between END2 promoter (SEQ ID NO:2) and PINII terminator (SEQ ID NO:3), reassembles into DsRed's (r) Expression casette (END2:DsRed (r): PINII)；

3) the full length nucleotide sequence of OsCYP704B2 gene, target gene OsCYP704B2 and its promoter and terminator As shown in SEQ ID NO:4, wherein the promoter sequence of OsCYP704B2 gene is as shown in SEQ ID NO:7, terminator sequence Column are as shown in SEQ ID NO:8, and the genomic dna sequence of OsCYP704B2 gene is as shown in SEQ ID NO:5, nucleotides sequence The protein amino acid sequence of coding is arranged as shown in SEQ ID NO:6；

4) its nucleotide sequence of expression casette PG47:ZM-BT1:ZM-AA1:IN2-1, target gene ZM-AA1(such as SEQ Shown in ID NO:9) opening code-reading frame be connected to its nucleotide sequence of promoter PG47(as shown in SEQ ID NO:10), transhipment Its nucleotide sequence of peptide ZM-BT1(is as shown in SEQ ID NO:11) downstream, its nucleotide sequence of terminator IN2-1(such as SEQ Shown in ID NO:12) upstream.

Specifically, the building process of carrier p7R is described in detail below:

The first step, expands pollen inactivated gene ZM-AA1 from the cDNA of maize calli and coding leads the ZM- of peptide BT1, amplification obtains PG47 promoter from corn gene group DNA.Wherein, for expanding the amplimer of ZM-AA1 are as follows:

F3:CGGTACCCGGGGATC CAAGGAAAAGACGTTATGCAG(SEQ ID NO:37), be in box Bgl II restriction enzyme site,

R3:AAGGTCGTCCGGGCGGCCTGCGGCCTGGTCCAGGCAC(SEQ ID NO:38), wherein underscore indicates Nucleotides sequence be classified as the overlap of 15bp；

For expanding the amplimer of ZM-TP are as follows:

F4:CGCCCGGACGACCTTGGGATCG(SEQ ID NO:39)；

R4:ATGGCGGCGACAATGGCAGTGAC(SEQ ID NO:40)；

For expanding the primer of PG47 promoter are as follows:

F5:CATTGTCGCCGCCATGGTGTCGTGATCGATGCTTTAT(SEQ ID NO:41),

R5:CGACTCTAGAGGATCTGCACCGGACACTGTCTGGTGG(SEQ ID NO:42), wherein underscore indicates Nucleotides sequence be classified as 15bp overlap).

Obtained ZM-AA1 gene will be expanded using the method for In-Fusion, ZM-BT1 leads peptide, and PG47 promoter three Segment is connected on the binary vector pCAMBIA1300 that BamHI digestion is opened, and obtains intermediate vector A.

Second step, artificial synthesized red fluorescent protein gene PINII-DsRed (r) sequence of PCR amplification, wherein artificial close At PINII-DsRed (r) sequence be as shown in SEQ ID NO:43, amplimer are as follows:

F1:ATTAACGCCGAATTGGCCGCATTCGCAAAACACACC(SEQ ID NO:44),

R1:CAAGAACTAGTAACCATGGCCTCCTCCGAGAACGTGA (SEQ ID NO:45), wherein italic underscore The nucleotides sequence of mark is classified as the overlap of 15bp.

END2 sequence, amplimer are expanded from corn gene group DNA are as follows:

F2:CTCGGAGGAGGCCATGGTTACTAGTTCTTGGGGGACG (SEQ ID NO:46),

R2:

CTTTTCCTTGAGATCGAATTCCTGCAGCCCGGGGGATCCAGCTTCGCTTAGTTTTTAGT(SEQ IDNO: 47) nucleotides sequence that, wherein italic underscore indicates is classified as the overlap of 15bp.

Use the method for In-Fusion that will expand obtained PIN II-DsRed (r) segment and from corn and for callus Tissue and seed (embryo and endosperm) specificity promoter END2 segment are connected into the intermediate vector A of Xmn I Yu Bgl II digestion simultaneously, Obtain intermediate vector B.

Third step, the nucleotide sequence of artificial synthesized IN2-1, sequence is as shown in SEQ ID NO:48.With EcoR I with The artificial synthesized IN2-1 sequence of BglII double digestion, wherein artificial synthesized IN2-1 sequence is as follows:

gacaaagcagcattagtccgttgatcggtggaagaccactcgtcagtgttgagttgaatgtt tgatcaataaaatacggcaatgctgtaagggttgttttttatgccattgataatacactgtactgttcagttgttg aactctatttcttagccatgccaagtgcttttcttattttgaataacattacagcaaaaagttgaaagacaaaaaa aaaaacccccgaacagagtgctttgggtcccaagctactttagactgtgttcggcgttccccctaaatttctcccc ctatatctcactcacttgtcacatcagcgttctctttcccctatatctccacgtcgacgcggccaaatcctgagga tctggtcttcctaaggacccgggatatcggacgggggatccactagttctagagcggccgggtaccgagctcgaat taattgtttaaactcgatAagggcaattccagcacactggcggccgttactagcga (SEQ ID NO:48), wherein underlined sequences are IN2-1 sequence, remaining sequence is that carrier connects region sequence, box mark Sequence respectively is: Bgl II, Asc I, Nru I and EcoR I restriction enzyme site, wherein five alkali after Bgl II restriction enzyme site In addition base has tri- restriction enzyme sites of Asc I, Nru I and EcoR I to connect on region sequence in carrier in IN2-1 sequence.Digestion Plasmid where artificial synthesized IN2-1 sequence, is connected into while with the intermediate vector B of EcoR I and Bgl II double digestion, obtains Intermediate vector C.

OsCYP704B2 gene is divided into two sections by the 4th step, is CYP1 and CYP2 respectively, and nucleotide sequence is respectively such as Shown in SEQ ID NO:60 and SEQ ID NO:61.From oryza sativa genomic dna, CYP1 and CYP2 is expanded respectively.

Wherein, the amplimer of CYP1 are as follows:

F6:CGATTGGTCGAACACGAGGTAGGCG (SEQ ID NO:49), interior box is Nru I restriction enzyme site；

R6:TCGAAGGACCGCACCGTGACCATG (SEQ ID NO:50), interior box is Sal I digestion position Point, the base of underscore mark be in order to distinguish the endogenous OsCYP704B2 gene order of rice, and improve expression efficiency and Three SNP being intentionally introduced into；

The amplimer of CYP2 are as follows:

F7:CATGGTCACGGTGCGGTCCTTCGA (SEQ ID NO:51), interior box is Sal I digestion position Point, underscore mark is and to improve expression efficiency and three SNP being intentionally introduced into distinguish the endogenous CYP sequence of rice；

R7:ATTGTTTAAACAGGTGGAAGACAAGGTGGTGAGG (SEQ ID NO:52) is in box Asc I restriction enzyme site

By obtained two amplified productions, after even the sequencing of T- carrier is correct, then Nru I and Sal I is used respectively, and Sal I and Asc I double digestion, while being connected into the intermediate vector C of Nru I and Asc I double digestion to get 7R carrier is arrived, also referred to as For p7R.

Embodiment 2: rice conversion

Plasmid p7R is transferred to Agrobacterium AGL0 bacterial strain using heat shock method, corotation is carried out to rice using agrobacterium-mediated transformation Change (Hiei, et al.Efficient transformation of rice mediated by Agrobacterium and Sequence analysis of the boundaries of the T-DNA.(1994) Plant J6 (2): 271-282, lead to It crosses reference to be incorporated into herein).Specific transformation receptor material is rice ms26 holandry infertility Mutants homozygous, the mutation Body is by radiation-induced gained, and mutation is to lead to (missing area by 3103bp missing (comprising OsCYP704B2 major part segment) Section physical location: ensembl plants oryza japonica group version64.6 (MSU6) chromosome3: 3,701,319-3,704,421).Large fragment deletion mutation keeps the probability of back mutation extremely low, therefore sterile character is stablized, thus It has ensured the stability of sterile line, has reduced hybrid seeding risk.Using Agrobacterium by plasmid p7R rice transformation acceptor material, benefit The red fluorescent protein for using DsRed (r) gene in carrier to encode is as selection markers, the callus fluorescent screening taken turns by 3-4 After cutting, differentiation obtains transgenic plant, which obtains 1000 plants of positive transgenic material or more by rice conversion, into After one step is by analyses such as insertion point, copy number, carrier framework pollutions, the pollen Inactivation Effect, the knot that are arrived in conjunction with field observation Real rate, seed segregation ratio etc. are as a result, therefrom preferably go out two transgenic events 7R-949D and 7R-1425D.

Embodiment 3: the pollen fertility detection of transformation event

Observation analysis discovery is carried out to two transgenic events 7R-949D and 7R-1425D obtained in embodiment 2, this It invents and apparent modal difference is not observed between obtained transgenic plant and non-transgenic control plant.

With transformation receptor strain corresponding Wild-type non-transgenic rice varieties force fortune round-grained rice 7 (it is fertile, hereinafter simply referred to as CK1) and the corresponding non-transgenic rice strain force of transgenic paddy rice transports No. 7 ms26 mutant of round-grained rice (infertility, hereinafter simply referred to as CK2) For control, rate detection can be contaminated by carrying out pollen.

In Rice Flowering advanced stage, single plant is respectively randomly selected from the transgenic paddy rice in crop field, CK1 and CK2 cell, each strain takes one Piece flower, every flower take 1 anther, are placed in glass slide center, and the I2-IK solution of a drop 1% is added dropwise, is discharged with tweezers and dissecting needle After pollen, covered is observed under the microscope, count can stained pollen number and pollen frequence (after Fig. 2 shows dyeing Fertile flower powder and not fertile pollen grain).Calculate separately transgenic paddy rice, CK1 and CK2 pollen can contaminate rate 3 repetitions of each material Average standard deviation.Under the premise of the normal fertile and CK2 of CK1 normal infertility, by Χ2 test, transgenosis water is analyzed The pollen of rice can contaminate rate, and whether there is or not significant differences with theoretical value (50%).

The results show that 7R-949D strain T2 for the pollen (T3 is for genotype) on plant to contaminate rate as shown in table 1 below. This test is carried out using pollen segregation ratio (3 duplicate average) of the I2-KI decoration method to 17 plant randomly selected Investigation, from table 1 it follows that the fertile rate of fertile control (CK1) is between 98.6%~100%；Infertility control (CK2) can Educating rate is 0；(freedom degree 1) is examined by Χ 2-, in 17 7R-949D single plants, except the chi-square value (7.454) of single plant 9 is micro- high Outside in critical value (3.81), fertile pollen and pollen sterile segregation ratio meet in the level of P≤0.05 in other 16 flowers The ratio of 1 ﹕ 1.

7R-1425D strain T2 for the pollen (T3 is for genotype) on plant to contaminate rate as shown in table 2.It randomly selects 17 plant calculate 3 duplicate averages.From Table 2, it can be seen that it is fertile control (CK1) fertile rate 95.0.1%~ Between 99.8%；Infertility control (CK2) fertile rate is 0；(freedom degree 1) is examined by Χ 2-, in 17 7R-949D single plants, is removed The chi-square value (6.426) of single plant 3 is higher than critical value (3.81) outside, fertile pollen and pollen sterile segregation ratio in other 16 flowers Example in the level of P≤0.05, meets the ratio of 1 ﹕ 1.

Should the result shows that, the T-DNA foreign gene in transformation event 7R-949D and 7R-1425D is in miscellaneous in each generation Conjunction state, therefore have half pollen without foreign gene (fertile), half contains foreign gene (infertility), and ZM-AA1 expression cassette The starch in pollen can be effectively decomposed, so that the pollen containing foreign gene be made to lose vigor, fertilizing ability is lost, causes Transgenic pollen inactivation.So that in above-mentioned two transformation event, the transgenic pollen containing ZM-AA1 gene is all lost for the design Living, cannot inseminate can also strictly prevent the bio-safeties problem such as genetic drift, the pollen of inactivation cannot with other plant around or Weeds pollination, thus transgenosis can not be by pollen dispersal into environment.

Table 1: transformant 7R-949D T₂For pollen I on plant₂- KI staining analysis

Note: X² _0.05(1)=3.81, * indicates to think to meet 1 ﹕, 1 ratio under the level of signifiance 0.05.

Table 2: transformant 7R-1425D T₂For pollen I2-KI staining analysis on plant

Note: X² _0.05(1)=3.81, * indicates to think to meet 1 ﹕, 1 ratio under the level of signifiance 0.05.

Embodiment 4: fluorescent seeds and non-fluorescence seed segregation ratio

Each 24 T2 by 7R-949D and 7R-1425D obtained in embodiment 2 are randomly selected for plant single plant, it is right The segregation ratio of tied fluorescence and non-fluorescence seed is investigated thereon, as a result as shown in table 3 below.Wherein, 2 value of Χ (df=1) Lower than critical value 3.81, shows to bear seeds on 2 each plant of transformant and meet 1:1 segregation ratio.Illustrate institute in the present invention Each element of the expression vector of offer is expressed well as a whole, i.e., conversion site is in miscellaneous in each generation of transformation event always Conjunction state, therefore have half pollen without foreign gene, half contains foreign gene, the half pollen inactivation containing foreign gene (losing fertilizing ability), so foreign gene only passes through oogamete and is transferred to the next generation, other one without containing foreign gene Half pollen can make transformant self-fertility, and the ratio of the tied fertile seed of fluorescence and non-fluorescence sterile seed is 1:1, fertile plant (having foreign gene), which is used as, keeps system, easily, continuously can produce sterile line by selfing and keep being infertility Strain (being free of transgene component) is used as the parent of hybrid seeding in production.

Table 3: tied fluorescent seeds and non-fluorescence seed number ratio are selfed on strain 7R-949D and 7R-1425D

Note: X² _0.05(1)=3.81, * indicates the level of signifiance below 0.05, it is believed that meets 1 ﹕, 1 ratio.

It can be seen from above-described embodiment 3 and embodiment 4 present invention realize its stablize initiative male sterible series of rice and Keep the goal of the invention of system.

Embodiment 5: the flanking sequence analysis of transformation event

Using transgenic rice lines 7R-949D obtained in embodiment 2 and the genomic DNA of 7R-1425D plant as mould Plate, p7R carrier and rice conversion receptor force fortune No. 7 ms26 mutant (infertility) plant of round-grained rice utilize TAIL- as negative control PCR amplification T-DNA flanking sequence, obtains the T-DNA flanking sequence of transformation event 7R-949D and 7R-1425D, and analyzes verifying The Integration Mode of T-DNA.The T-DNA Integration Mode speculated using regular-PCR verifying, and analyze foreign gene whether be inserted into it is known Inside rice endogenous gene.

Primer information used in analytical methods is shown in Table 4.

The primer information used in table 4PCR amplification procedure

Primer	Primer sequence (5 ' -3 ', SEQ ID NO :)
		SP1	ATAATGTGGGCATCAAAGTTGTGTG(24)
SP2	CTTATCCTAAATGAATGTCACGTGTCTT(25)
		SP3	TAGGTGTGTTTTGCGAATGCGGC(26)
LAD1-1	ACGATGGACTCCAGAGCVNVNNNGGAA(27)
		AC1	ACGATGGACTCCAGAG(28)
7RB-1	TCGTGACTGGGAAAACCCTGGC(29)
		7RB-2	GCTGGCGTAATAGCGAAGAGGC(30)
7RB-3	TCAGATTGTCGTTTCCCGCCTTCA(31)
		A949B-L-1	GTGGATGAAAGCCTTCGTTAC(32)

A949B-R	TTCCACCACACACCAAAACCA(33)
		A1425LB-2	TAAAGAAGGCTCGCAAGTGTG(34)
A1425RB-2	CATCCTAGTCATTGGGTTGGG(35)

The oryza sativa genomic dna of extraction is suitably diluted, measure and records it in the UV Absorption of 260nm and 280nm Rate is equivalent to 50 μ g/mL DNA concentrations with an OD260 value to calculate the DNA concentration of purifying.DNA solution OD260/OD280's Ratio is between 1.7~2.0.4 DEG C save use in one week.

Hot asymmetric interlaced PCR (the THERMAL ASYMMETRIC improved designed according to Liu Yaoguang et al. INTERLACED PCR TAIL-PCR) (Liu et al., Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asym-metric Interlaced PCR, (1998) Plant Molecular Biology Reporter16 (2): 175-181, by referring to general It is incorporated herein), on the T-DNA of external source insertion 3 nested special primers of design (SPECIALPRIMER, respectively SP1, SP2, SP3), combine SP1 and arbitrary degenerate primer (LONG ARBITRARYDEGENERATE PRIMER, LAD), and SP2 and SP3 is combined with anchor primer AC1 respectively, using genomic DNA as template, is designed according to primer length and specific difference asymmetric Temperature cycles, carry out TAIL-PCR.Wherein, reaction process is as shown in table 5.

Table 5TAIL-PCR experiment flow

The product of the wheel of TAIL-PCR second and third round is clicked and entered sample cell, 120V constant pressure by the Ago-Gel of preparation 1% Electrophoresis 20 minutes, gel is observed in the UV lamp, and the product of size nesting is cut, use Ago-Gel QIAquick Gel Extraction Kit (TIANGEN, Beijing) recycles PCR product.Using T4 ligase (New England Biolabs, the U.S.) by purification and recovery PCR product be connected in T- carrier (Promega, the U.S.), the system in 16 DEG C reaction overnight.Linked system is as shown in table 6.

Table 6T- carrier linked system

Reagent	Volume
		T4 ligase 10X Buffer	2μl
T4 ligase	0.4μl
		PCR product	2μl
pGEM-T	0.4μl
		Deionized water	15.2μl

The overnight connection liquid of above-mentioned 16 DEG C reactions is converted into Escherichia coli, the single colonie that picking obtains, in the LB of the benzyl containing ammonia DNA sequencing is extracted in 37 DEG C shaken cultivation (150rpm) 5 hours in culture solution.

According to TAIL-PCR's as a result, flanking sequence design primer according to the insertion position T-DNA, with T-DNA inner primer Combination carries out PCR amplification by template of genomic DNA.PCR reaction system is as shown in table 7.

Table 7PCR reaction system (20 μ l system)

Reagent	Volume
		10X buffer	2μl
10mM dNTP	0.5μl

DMSO	0.133μl
		Taq enzyme	0.25μl
DNA profiling	1μl
		Deionized water	16.117μl

PCR amplification condition is set according to different primers, and PCR product is run into Ago-Gel detection.

The flanking sequence of acquisition is subjected to sequencing analysis, and with database (MSU Rice Genome Annotation Project Release7, issuing time on October 31st, 2011, ftp: //ftp.plantbiology.msu.edu/pub/ data/Eukaryotic_Projects/o_sativa/annotation_dbs/pseudomolecules/version_ 7.0/) Rice Genome Sequence is compared in, finds in 7R-949D transformation event, T-DNA is inserted in rice the 3rd dye At the nearly centromere of colour solid galianconism, physical location Chr3:14,746,015-14,746,027, it is not inserted into the endogenous base of known rice Because of code area, the initiation codon 4411bp away from upstream gene LOC_Os03g25760, away from downstream gene LOC_Os03g25770 Terminator codon 5804bp(Fig. 3, Fig. 4).T-DNA integration causes genome sequence at integration site to lack 11 bases, lacks Out-of-sequence column are 5 ' GGGGGTCGGTG3 ' as shown in SEQ ID NO:63, which does not destroy rice endogenous gene Area.

In 7R-1425D transformation event, T-DNA is inserted in No. 1 chromosome long arm distal end of rice, and physical location is Chr1:42,215,016-42,215,095, and it is not inserted into known rice endogenous gene area, away from upstream gene LOC_ Os01g72760 terminator codon 1343bp, away from downstream gene LOC_Os01g72780 initiation codon 1953bp(Fig. 5, Fig. 6). T-DNA integration causes genome sequence to lack 78 bases, and as shown in SEQ ID NO:62, which does not break deletion sequence Bad rice endogenous gene area.

In two transformation events, T-DNA is not inserted into inside known rice endogenous gene.To external source T-DNA and insert The engaging zones for entering the genomic DNA of position carry out PCR amplification to verify the insertion position external source T-DNA and speculate that T-DNA is integrated As a result mode further demonstrates T-DNA insertion that is, to carry out PCR amplification between side sequence and T-DNA lateral order column for target sequence The correctness in site, and show that double copy unit points that 7R-949D is differential concatenation are integrated, result is as shown in figure 4, insert The specific nucleotide sequence of the 5 ' ends of the external source T-DNA entered and 3 ' end flanking sequences is respectively such as SEQ ID NO:13 and SEQ ID Shown in NO:14；And the T-DNA of 7R-1425D is single copy insertion, result as shown in fig. 6, the external source T-DNA of insertion 5 ' ends Specific nucleotide sequence with 3 ' end flanking sequences is respectively as shown in SEQ ID NO:15 and SEQ ID NO:16.

Further, the complete external source T- of insertion transformation event 7R-949D and 7R-1425D is obtained by sequencing analysis DNA sequence dna, the particular sequence is respectively as shown in SEQ ID NO:53 and SEQ ID NO:54.By in two transformation events of analysis The sequencing result of external source T-DNA segment, it was demonstrated that the external source T- copied there are two being integrated in the genome of transformation event 7R-949D DNA, and differential concatenation, one of copy is identical as carrier, the PG47 promoter deletion 1965bp in another copy, should Missing does not influence the natural biological function of each element and expression cassette, is then only integrated in the genome of transformation event 7R-1425D The T-DNA of one complete copy.

Embodiment 6: exogenous origin gene integrator stability analysis in transformation event

In order to verify the copy number and integrality of external source gene insertion in above-mentioned transformation event, the present invention is used The method of Southern blot marking analysis analyzes above-mentioned transformation event.

According to the T-DNA sequence design probe in carrier, wherein for detecting target gene in transformation event 7R-949D The probe sequence of (including OsCYP704B2 and ZM-AA1 expression cassette) detects color sorting gene DsRed as shown in SEQ ID NO:55 (r) probe sequence is as shown in SEQ ID NO:56.Probe binding site in transformation event 7R-949D on external source T-DNA and Restriction enzyme site is as shown in fig. 7, wherein Fig. 7 A shows probe sequence position and Hind III digestion site on target gene；Fig. 7 B Show I restriction enzyme site of probe sequence position and EcoR on color sorting gene.

Probe sequence for detecting OsCYP704B2 in transformation event 7R-1425D is detected as shown in SEQ ID NO:57 The probe sequence of pollen inactivated gene ZM-AA1 detects the probe sequence of color sorting gene DsRed (r) as shown in SEQ ID NO:58 As shown in SEQ ID NO:59.Probe binding site and restriction enzyme site such as Fig. 8 in transformation event 7R-1425D on external source T-DNA With shown in Fig. 9, wherein Fig. 8 shows transformation event 7R-1425D using OsCYP704B2 as probe, after HindIII digestion, carries out Expected clip size caused by Southern blot；Fig. 9 show transformation event 7R-1425D respectively with Zm-AA1 and DsRed (r) is probe, after EcoR I digestion, carries out expected clip size caused by Southern blot.

It is transported with T2, T3 and T4 of transgenic paddy rice strain 7R-949D and 7R-1425D for plant and transformation receptor material force No. 7 ms26/ms26 mutant of round-grained rice are experimental material, carry out the analysis of the Southern blot marking by experiment flow below.

1, DNA is extracted

It is operated by People's Republic of China (PRC) agricultural industry criteria NY/T674, extracts oryza sativa genomic dna.DNA is suitably dilute It releases, measure and records it in the uv absorption rate of 260nm and 280nm, it is dense that 50 μ g/mL DNA are equivalent to an OD260 value It spends to calculate the DNA concentration of purifying.The ratio of DNA solution OD260/OD280 is between 1.7~2.0.It will according to the concentration measured DNA solution is diluted to 100ng/ μ L, and 4 DEG C save use in one week.

2, digoxin labelled probe (random priming)

1ug (at least 300ng) template DNA, is diluted to 16ul with sterile ddH2O；

Boiling water bath boils 10min, is immediately placed in ice；

It mixes DIG-High Prime(bottles 1), adds 4ul into denatured DNA, mix, be slightly centrifuged；

37 DEG C are reacted overnight (about 20 hours)；

2ul0.2M EDTA(Ph8.0 is added) or 65 DEG C of 10min termination reactions.

3, detection probe efficiency

After control probe and a series of dilutions of probe mark progress, directly put on film, by standard detection come Determine the labeling effciency of target probe.

4, electrophoresis and transferring film

(1) 1% Ago-Gel is prepared, constant pressure 45V electrophoresis is stayed overnight；

(2) bromjophenol blue away from well 6-8cm or so when stop electrophoresis.Well and extra glue are cut away, the lower left corner is cut away It marks；

(3) gel is immersed into 200ml, carries out depurination treatment about 5-10min in 0.25M HCl；

(4) it after rinsing gel taking-up once in deionized water, immerses in denaturing liquid, 2 × 15min；

(5) gel is taken out, rinses, is then immersed in neutralizer in deionized water, 2 × 15min；

(6) after rinsing gel taking-up once in deionized water, capillary transfer is carried out；

(7) the capillary transfer of DNA: contained in big culture dish 20 × SSC → restocking glass plate → glass plate upper berth thickness filter paper → Catch up with bubble → by gel well be placed on filter paper bridge downward → around lived with Parafilm membrane cover → glue on the big nylon such as put Film → catch up with bubble → put the big filter paper such as four layers and film on film → catch up with bubble → above putting the paper handkerchief of 10cm thickness → put glass plate → on Press 500g weight；

(8) capillary transfer for 24 hours, is taken out film and is washed in 2 × SSC, be sandwiched in filter paper, be sandwiched in filter paper, 254nmUV, UV crosslinking 3min.

5, hybridize

(1) film immersion prehybridization: is preheating to (10ml/100cm in (52 DEG C) the DIG Easy Hyb of hybridization temperature² Film), 52 DEG C, 60rpm, prehybridization 1h in hybridization case；

(2) DNA probe (about 25ng/ml DIG Easy Hyb) is put on ice immediately after 100 DEG C of denaturation 10min, Cooling 10min；

(3) DNA probe of denaturation is added to (3.5ml/100cm in (52 DEG C) the DIG Easy Hyb of preheating²Film), softly It mixes, avoids foam；

(4) prehybridization solution is outwelled, hybridization solution is poured into hybridization bottle, 52 DEG C, 60rpm, is hybridized 12-16 hours；

(5) hybridization terminates, and recycles hybridization solution, and -20 DEG C can be reserved for 1 year, then 68 DEG C of heating 10min of used time are denaturalized spy again Needle.

6, film is washed

1) low preciseness (with high salt, low temperature): 2 sufficient × SSC+0.1%SDS, room temperature 60rpm wash 2 × 15min；

2) high preciseness (less salt, high temperature): 0.5 × SSC+0.1%SDS(is preheating to wash temperature), 60rpm washing 2 × 15min。

Note: if probe > 150bp and G/C% higher, film should be washed at 68 DEG C；When shorter than 100bp, wash temperature is the same as miscellaneous Hand over temperature.

7, it detects

Applied chemistry shines detection method, and all operations are all carried out in room temperature.

1) after hybridization terminates and washes film, the of short duration flushing membrane about 1-5min in Washing buffer；

2) 30min(jog is closed in 80ml Blocking solution)；

3) 40min is reacted in 20ml Antibody solution；

4) transfer membrane enters new container, is washed twice with Washing buffer, each 15min；

5) 2-5min is balanced in 20ml Detection buffer.

6) film DNA is carefully placed into up in hybridization bag, draws ready-to-use(Kit bottles of 1ml CSPD 5) uniformly It is applied on film, immediately covers on the upper layer of hybridization bag on film, so that substrate is uniformly covered with film surface, and bubble is avoided to generate, React at room temperature 5min；

7) surplus liquid is squeezed out, the edge of hybridization bag is sealed and (prevents film dry)；

8) film is placed on 37 DEG C, 10min, to strengthen luminescence-producing reaction；

9) X- film 15-25min is exposed, result is observed.

The T of conclusion 1, transformant 7R-949D₂、T₃And T₄For the Southern of exogenous origin gene integrator stability in plant Blot analyzes result

Using transformation receptor force fortune No. 7 ms26/ms26 mutant DNA of round-grained rice as negative control, Plasmid DNA is added to military fortune Positive control is used as in No. 7 wild type gene group DNA of round-grained rice, probe can smoothly be combined under this hybridization conditions with target sequence.

The integration stability result of target gene is analyzed

With Hind III digestion transformant 7R-949D T2, T3 and T4 for plant genomic DNA, designed according to target gene Probe carries out Southern blot.As the result is shown strain T2, T3 and T4 for have in the single plant of plant~5.6kb and~ The signal strips band (Figure 10) of 7.9kb, actual observation segment and clip size (i.e. 5574bp and 7921bp) phase expected in transformant It accords with (table 8), shows to be 2 copies.Stripe size is identical between generation-inter-, and copy number is consistent, shows the purpose of transformant 7R-949D Gene T2, T3 and T4 instead of between stablize heredity.

The analysis of color sorting gene integration stability result

With EcoR I digestion transformant 7R-949D T2, T3 and T4 for plant genomic DNA, set according to color sorting gene order It counts probe and carries out Southern blot detection.The results show that T2, T3 and T4 of the strain in three single plants of plant for having The signal strips band (Figure 11) of~18kb and~8.4kb.Expected clip size (i.e. 18184bp in actual observation segment and transformant Be consistent (table 8) with 8425bp).3 generation intermolecular hybrid signal strips algebra mesh are identical, and stripe size is consistent, shows transformant 7R-949D's Color sorting gene T2, T3 and T4 instead of between stablize heredity.

In conclusion according to T-DNA sequence, insertion point flanking sequence, probe location and digestion on transformant 7R-949D Site can predict hybridized fragment size.Actual observation is compared with prediction, to confirm the copy number of foreign gene.From Table 8 is as can be seen that the actual observation segment of all hybridization is consistent with corresponding predicted segment size.Therefore, foreign gene is in 7R- T2, T3 and T4 of 949D instead of between stable integration, and keep 2 copies it is constant.

T2, T3 and T4 of 8 transformant 7R-949D of table for foreign gene in plant Southern blot predicted segment and Actual observation clip size

Probe is designed according to objective gene sequence and color sorting gene order, T2, T3 and T4 generation of transformant 7R-949D is planted Pnca gene group DNA carries out Southern blot, the results show that foreign gene is 2 copies, the number phase of hybridising band between 3 generations Together, stripe size is consistent, shows that the foreign gene of the transformant stablizes heredity between three generations.

Conclusion 2, transformant 7R-1425D T2, T3 and T4 for foreign gene in plant the Southern for integrating stability Blot analysis

Using transformation receptor force fortune No. 7 ms26/ms26 mutant DNA of round-grained rice as negative control, Plasmid DNA is added to military fortune Positive control is used as in No. 7 wild type gene group DNA of round-grained rice, probe can smoothly be combined under this hybridization conditions with target sequence.

The integration stability result of OsCYP704B2 gene is analyzed

With Hind III digestion transformant 7R-1425D T2, T3 and T4 for plant genomic DNA, according to OsCYP704B2 Gene designs probe and carries out Southern blot.As the result is shown T2, T3 and T4 of the strain for have in the single plant of plant~ The signal strips band (Figure 12) of 8.3kb.Actual observation segment is consistent (table 9) with clip size (i.e. 8348bp) expected in transformant, Show to be 1 copy.Stripe size is identical between generation-inter-, and copy number is consistent, shows the OsCYP704B2 of transformant 7R-1425D Gene T2, T3 and T4 instead of between stablize heredity.

The integration stability result of ZM-AA1 gene is analyzed

With EcoR I digestion transformant 7R-1425D T2, T3 and T4 for plant genomic DNA, set according to ZM-AA1 gene It counts probe and carries out Southern blot detection.As the result is shown T2, T3 and T4 of the strain for have in three single plants of plant~ The signal strips band (Figure 13) of 5.9kb, actual observation segment are consistent (table 9) with clip size (i.e. 5934bp) expected in transformant, Show to be 1 copy.3 generation intermolecular hybrid signal strips algebra mesh are identical, and stripe size is consistent, shows transformant 7R-1425D's ZM-AA1 gene T2, T3 and T4 instead of between stablize heredity.

The integration stability result of DsRed (r) gene is analyzed

With EcoR I digestion transformant 7R-1425D T2, T3 and T4 for plant genomic DNA, according to DsRed (r) gene Sequence design probe carries out Southern blot detection.T2, T3 and T4 of the strain are in three single plants of plant as the result is shown There is~signal strips the band (Figure 14) of 4.8kb.Actual observation segment is consistent with clip size (i.e. 4824bp) expected in transformant (table 9) is 1 copy.3 generation intermolecular hybrid signal strips algebra mesh are identical, and stripe size is consistent, shows transformant 7R-1425D's DsRed (r) gene T2, T3 and T4 instead of between stablize heredity.

In conclusion according to T-DNA sequence, insertion point flanking sequence, probe location and enzyme on transformant 7R-1425D Enzyme site predicts hybridized fragment size.Actual observation is compared with prediction, to confirm the copy number of foreign gene.Under Table 9 is as can be seen that the actual observation segment of all hybridization is consistent with corresponding predicted segment size.Therefore, foreign gene is in 7R- T2, T3 and T4 of 1425D instead of between stable integration, and keep 1 copy it is constant.

T2, T3 and T4 of 9 transformant 7R-1425D of table for foreign gene in plant Southern blot predicted segment and Actual observation clip size

Design probe according to OsCYP704B2, Zm-AA1 and DsRed (r) gene order, to the T2 of transformant 7R-1425, T3 and T4 carries out Southern blot for plant genomic DNA, the results show that foreign gene is 1 copy, hybridization between 3 generations The number of band is identical, and stripe size is consistent, shows that the foreign gene of the transformant stablizes heredity between three generations.

Embodiment 7: the expression analysis (RT-PCR detection) of foreign gene in transformation event

With transgenic paddy rice strain T2 for seedling stage root, seedling stage stem, seedling stage leaf, clever flower primordium idiophase children's fringe (P3 phase), flower Powder mother cell Meiosis children fringe (P6 phase), pollen maturation phase children's fringe (P8 phase), Grain Ripening period seed cDNA be Template respectively expands target gene OsCYP704B2, Zm-AA1 and DsRed (r), and goal in research gene is in rice difference Expression in growth phase different tissues.

Materials period, tissue and detection target gene are shown in Table 10.

100mg sample liquid feeding nitrogen is taken to be ground into powdery in mortar, rapidly plus the every 100mg of Trizol(adds 1ml) it grinds again It is milled to even tissue, is then transferred to lapping liquid centrifuge tube (every pipe 1ml), 0.2ml chloroform is added in every pipe, and fierceness is shaken, directly To mixing.It is centrifuged 6min, 14000rpm, 400ul supernatant is taken to add 500ul isopropanol, slightly shake up up and down to new centrifuge tube, in It is stored at room temperature 2min, room temperature is centrifuged 5min, 14000rpm, removes supernatant, adds 500ul75% ethyl alcohol (DEPC processing) washing precipitating 2 Secondary, room temperature is centrifuged 2min, and 12000rpm, naturally dry (general 15min) is afterwards plus 40 μ l deionized waters (DEPC processing) dissolves RNA Precipitating.Finally use DNase enzymic digestion half an hour.Reverse transcription according to Fermentas RevertAidTM First Strand The operating procedure of cDNA Synthesis Kit, by RNA reverse transcription at cDNA.

Sample phase, position and the gene detected of 10 test material of table

Primer and amplified production

It is set using the expressed sequence of OsCYP704B2 gene, DsRed (r) gene, Zm-AA1 gene and Actin gene as template It counts primer (table 11).Across the introne design of the primer of Actin gene and OsCYP704B2 gene, the cDNA of the two genes are Template amplification product length is theoretically smaller than with the length for the amplified production that genomic DNA (gDNA) is template.

11 target gene of table and reference gene qualitative PCR primer information

Note: base number is corresponding primer and template PCR amplifications primer size in bracket

PCR amplification

The Establishing (table 12) that PCR system is provided according to Taq enzyme specification.Amplification program is equal are as follows: 94 DEG C of 5min；94℃ 0.5min, 60 DEG C of 0.5min, 72 DEG C of 1min, 30 circulations；72℃10min.After PCR amplification, each reaction system takes 10 μ L to expand Product is detected for 1.5% agarose gel electrophoresis.

Table 12PCR amplification system

The expression pattern of 3 target genes in 7R-949D transformation event

7R-949D T2 is extracted respectively for plant and the military fortune No. 7 ms26/ms26(mutant of round-grained rice of control), military fortune round-grained rice 7 it is (wild Raw type) seedling stage root, seedling stage stem, seedling stage leaf, P3 phase, P6 phase, the RNA of P8 phase and Grain Ripening period seed and reverse transcription obtain CDNA is obtained, is template to target gene OsCYP704B2, Zm-AA1, DsRed (r) and reference gene Actin points using these cDNA (Figure 15) is not expanded.

(1) without amplified band in blank control, show that PCR amplification system is polluted without target sequence；

(2) positive control is using transgenic line genomic DNA as template, can amplify purpose band, and size and pre- Phase is consistent, and shows that PCR amplification system can effectively expand target sequence；

(3) using the cDNA of above-mentioned transgenic line material as template, the reference gene Actin piece of about 554bp is amplified The target gene OsCYP704B2 segment of section and 344bp, less than (size is distinguished using genomic DNA as the pcr amplification product of template For 803bp and 432bp), show that RNA is extracted and reverse transcription is successful, and without DNA pollution.

(4) using the cDNA of round-grained rice 7 (wild type) different times different tissues of military fortune as template, in only P6 phase children fringe cDNA Amplify the OsCYP704B2 segment of about 344bp size.Show rice endogenous gene OsCYP704B2 in P6 phase specificity table It reaches.

(5) No. 7 ms26/ms26(mutant of round-grained rice are transported using military) cDNA of different times different tissues as template, do not expand to OsCYP704B2 gene product shows that mutation causes the expression deletion of the endogenous OsCYP704B2 gene of rice.

(6) target gene is expanded as template using the cDNA of 7R-949D different times different tissues, in P6 phase children fringe CDNA amplifies the OsCYP704B2 segment of about 344bp size, amplifies about 882bp size in P8 phase children fringe cDNA ZmAA1 segment amplifies DsRed (r) segment of general 365bp size in Grain Ripening phase in period seed；Experimental result card Bright OsCYP704B2 gene is specific expressed in the P6 phase, and Zm-AA1 gene is specific expressed in the P8 phase, and DsRed (r) gene is being planted It is specific expressed in son.

The expression pattern of 3 target genes of 7R-1425D transformant

Extract 7R-1425D T2 respectively and transport No. 7 ms26/ms26(mutant of round-grained rice for plant and control material force), military fortune round-grained rice 7 Seedling stage root, seedling stage stem, the seedling stage leaf, P3 phase, P6 phase, P8 of number (wild type)) and Grain Ripening period seed RNA and reversion Record obtains cDNA, is template to target gene OsCYP704B2, Zm-AA1, DsRed (r) and reference gene using these cDNA Actin is expanded (Figure 16) respectively.

(1) without amplified band in blank control, show that PCR amplification system is polluted without target sequence；

(2) positive control is using transgenic line genomic DNA as template, can amplify purpose band, and size and pre- Phase is consistent, and shows that PCR amplification system can effectively expand target sequence；

(3) using the cDNA of above-mentioned transgenic line material as template, the reference gene Actin piece of about 554bp is amplified The target gene OsCYP704B2 segment of section and 344bp, less than (size is distinguished using genomic DNA as the pcr amplification product of template For 803bp and 432bp), show that RNA is extracted and reverse transcription is successful, and without DNA pollution.

(4) using the cDNA of round-grained rice 7 (wild type) different times different tissues of military fortune as template, in only P6 phase children fringe cDNA Amplify the OsCYP704B2 segment of about 344bp size.Show that rice endogenous gene OsCYP704B2 is special in the rice P6 phase Property expression.

(5) No. 7 ms26/ms26(mutant of round-grained rice are transported using military) cDNA of different times different tissues as template, do not expand to OsCYP704B2 gene product shows that mutation causes the expression deletion of the endogenous OsCYP704B2 gene of rice.

(6) target gene is expanded as template using the cDNA of 7R-1425D different times different tissues, in the P6 phase Young fringe cDNA amplifies the OsCYP704B2 segment of about 344bp size, and it is big to amplify about 882bp in the young fringe cDNA of P8 phase Small ZmAA1 segment amplifies DsRed (r) segment of general 365bp size in Grain Ripening phase in period seed；Experiment knot Fruit proves that OsCYP704B2 gene is specific expressed in the P6 phase, and Zm-AA1 gene is specific expressed in the P8 phase, DsRed (r) gene It is specific expressed in seed.

Industrial applicibility

Construct of the invention can be effectively applied to male sterible series of rice and keep the building of system, and then obtain Stable fertility male sterible series of rice and keep system that can be efficiently applied to the production of hybrid seed, so as to obtain Safety, good hybrid rice seeds.

Although a specific embodiment of the invention has obtained detailed description, it will be understood to those of skill in the art that.Root According to all introductions having disclosed, those details can be carry out various modifications and be replaced, these change in guarantor of the invention Within the scope of shield.Full scope of the invention is given by the appended claims and any equivalents thereof.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " illustrative examples ", The description of " example ", " specific example " or " some examples " etc. means specific features described in conjunction with this embodiment or example, knot Structure, material or feature are included at least one embodiment or example of the invention.In the present specification, to above-mentioned term Schematic representation may not refer to the same embodiment or example.Moreover, specific features, structure, material or the spy of description Point can be combined in any suitable manner in any one or more of the embodiments or examples.

Claims (10)

1. a kind of rice conversion event SPT-7R-949D, which is characterized in that the transformation event contains an external source insertion sequence Column, the nucleotide sequence of the external source insetion sequence as shown in SEQ ID NO:53, the external source insetion sequence 5 ' end with it is interior Source genomic DNA constitute catenation sequence as shown in SEQ ID NO:13 or 17, the external source insetion sequence 3 ' end with it is endogenous The catenation sequence that genomic DNA is constituted is as shown in SEQ ID NO:14 or 18.

2. the method for identifying event SPT-7R-949D described in the claim 1 in biological sample comprising:

A) the first probe or the of specific recognition external source T-DNA insetion sequence SEQ ID NO:53 or its complementary series is designed One primer；With

B) the second probe or the second primer detection of the shoulder portion sequence of specific recognition SEQ ID NO:13 or 14 are designed The special area of SPT-7R-949D；

Wherein the first and second probes or primer mononucleotide segment detected are less than 100,000bp.

It further comprise carrying out polymerase chain reactions using at least two primers 3. method as claimed in claim 2, from described The DNA segment of amplification identification SPT-7R-949D event in biological sample, wherein the first primer specific recognition external source T-DNA insetion sequence SEQ ID NO:53 or its complementary series, the described second primer specificity identification SEQ ID NO:13 or 14 shoulder portion sequence.

4. the method for event SPT-7R-949D described in the claim 1 in a kind of detection biological sample, comprising:

(a) from the extraction from biological material DNA sample；

(b) DNA primer pair is provided, wherein the first primer specific recognition external source T-DNA insetion sequence SEQ ID NO:53 or its complementary series, the second primer specificity identify the shoulder portion sequence of SEQ ID NO:13 or 14；

(c) DNA amplification reaction condition is provided；

(d) DNA amplification reaction is carried out, DNA cloning is obtained；With

(e) the DNA cloning product is detected, wherein detection of DNA cloning in the DNA amplified reaction shows The presence of event SPT-7R-949D.

5. method as claimed in claim 4, wherein the DNA cloning attached bag include it is all or part of selected from SEQ ID NO: 13, the detection of 14,17,18 or 53 sequence, DNA cloning shows the presence of event SPT-7R-949D.

6. the method for external source insertion DNA molecular, the method packet in event SPT-7R-949D described in a kind of detection claim 1 It includes: the sample comprising DNA is contacted with polynucleotide probes, the polynucleotide probes are under stringent hybridisation conditions, Ke Yite The DNA of opposite sex detection SPT-7R-949D transformation event, shows the presence of SPT-7R-949D event, which is characterized in that the spy Needle is selected from partial sequence shown in SEQ ID NO:53.

7. method of claim 6, wherein the nucleotide sequence such as SEQ ID NO:55 of the polynucleotide probes or Shown in 56.

8. application of the transformation event described in claim 1 in breeding.

9. for identifying the primer pair or probe pair of event SPT-7R-949D described in the claim 1 in biological sample, packet Contain:

A) the first probe or the first primer of selectively targeted First ray, wherein the First ray being targeted is located at SEQ In the T-DNA insetion sequence of ID NO:53；And

B) the second probe or the second primer of selectively targeted second sequence, wherein second sequence being targeted is included in Flank region in sequence selected from SEQ ID NO:13,14,17 and 18；

Wherein first and second target sequence is detected in the single nucleic acid fragment less than 100,000 base-pairs.

10. the kit of transformation event SPT-7R-949D described in detection claim 1 a kind of, it includes the primers of claim 9 Pair or probe pair.