CN101092624B - Method for constructing gene of raining dual resistances of potato on PVX virus and PVY virus - Google Patents
Method for constructing gene of raining dual resistances of potato on PVX virus and PVY virus Download PDFInfo
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Abstract
This invention relates to a method for constructing a gene, especially a method for constructing a gene for improving both PVX resistance and PVY resistance of potato, which overcomes a plurality of problems in the improvement of the double resistance to the two viruses of potato. The method comprises the following steps: connecting a conserved gene segment on a PVX coat protein gene complete sequence cp and the conserved gene segment on a PVY replicase gene complete sequence NIb having the same restriction enzyme sites to form an artificial fusion gene cn; respectively connecting the artificial fusion gene cn to two sides of an intron in a manner of forward and backward to construct a backward repeat sequence of the artificial fusion gene, and two ends of the backward repeat sequence of the artificial fusion gene cn respectively being connected with a promotor and a terminator. When the backward repeat sequence of the fusion gene is transferred into potato, both PVX resistance and PVY resistance of the potato are improved. The resistance reaches high resistance even immune level and suits for production application.
Description
Technical field
The present invention relates to a kind of construction process of gene, be specially a kind of construction process that improves potato to the gene of PVX virus and the two resistances of PVY virus.
Background technology
Potato is important grain dish dual-purpose crop, in the course of cultivation, because the intrusion of virus has destroyed the normal physiological function of plant, cause plant strain growth decline, plant to become short, blade face shrinkage, yellowish green alternate embedding spot appears in blade, even the vein necrosis, whole compound leaf comes off, tie that the potato piece is little, proterties is poor, have a strong impact on yield and quality, Here it is say usually the potato degeneration phenomenon.The viral species that can infect potato has kind more than 30, with just the having of potato name 16 kinds (Xie Lianhui etc., 1999).In China, the virus that infects potato mainly is potato virus X (PVX), marmor upsilon (PVY), marmor solani (PVA), potato virus S (PVS), corium solani (PLRV) etc.These viral decapacitation are infected separately outside the potato plant, sometimes can also with compound the infecting of other virus, promptly at the virus infection that has on the same plant more than 2 kinds or 2 kinds, viral and the compound of PVY virus of the modal PVX of being infected in productions.Can cause than PVX virus or PVY virus after PVX virus and compound the infecting of PVY virus and infect more serious shrinkage flower leaf type degeneration separately, significantly improve when at this moment the content of PVX virus in plant infects more separately, even improve 10 times.PVX virus and PVY virus is compound infect the shrinkage flower leaf type that causes degenerate be the most common in the production, also be a kind of the most serious degenerated form, have a strong impact on the yield and quality of potato.Potato stem apex detoxification tissue culture technique can be produced the very low potato seed of viral level, but cost high workload amount is big, need special detoxication and tissue culture facility, potato seed after the detoxification can be subjected to tillage control measure and infecting of aphid and infective virus again in the course of cultivation, cause 1 year after output can seriously descend again.The potato vegetative propagation is again the autotetraploid crop, and with conventional breeding method improvement kind, the process complexity takes longer, and utilizing genetically engineered to cultivate antiviral potato is the effective way that fundamentally solves potato degeneration.
Along with the continuous development of art technology, 1985, Sanford and Johnstone proposed the notion of cause of disease deutero-resistance (PDR).1986, Beachy research group confirms that first the transgenic plant of expressing tobacco mosaic virus (TMV) (TMV) coat protein produce resistance to its donor virus TMV, occur many virogenes that utilize subsequently and carried out antiviral genetically modified report, institute's transgenosis comprises the complete sequence or the partial sequence of virus capsid protein, floating preteins, replicase protein gene, utilizes the gene of viral source to obtain the important channel that resistant transgenic plants has become the plant virus resistance gene engineering.Potato often is subjected to infecting of two or more viruses during growing, as PVX and PVY and PVY and usually mixed infection of PLRV, the living effect of the association of two kinds of viruses (synergism) has aggravated the hazard rating to potato.Utilizing the gene acquisition multiresistance transgenic Rhizoma Solani tuber osi of viral source is a big focus of potato antiviral gene engineering, as change the two valency coat protein genes of PVX and PVY or change PVY and the potato plant of the two valency coat protein genes of PLRV hold concurrently anti-PVX and PVY or PVY and two kinds of viruses of PLRV (Lawson et al, 1990; Song Yanru etc., 1994; Zhang Heling etc., 1996,1997).The transgenic Rhizoma Solani tuber osi that these research and utilization virogenes or gene fragment obtain is all being received certain effect aspect prevention or the reduction virus infection, but be difficult to reach immune level, the anti-diseased plant rate of the resulting transgenic Rhizoma Solani tuber osi of this method is lower, and resistance is delayed onset or alleviates occurring degree, disease-resistant level is not high, is difficult to use in production; And the RNA that transcribes out of the coat protein gene of above-mentioned two target virals can be kept perfectly even further translates into protein.Homologous recombination might take place with intravital other the viral RNA of invaded plants in the RNA that this transgenosis is transcribed out, and the protein that translates might the allos packing take place and produce new virus with intravital other the viral RNA of invaded plants.
Newly-developed double-stranded RNA rapidly disturbs (RNAi, RNA interference) technology to provide new approaches for the plant virus resistance transgenic engineering.The transcription product of normal gene can not form double-stranded RNA because of no complementary sequence in the organism; if double-stranded RNA; biological intravital nuclease will be degraded into it siRNA fragment (siRNA of 21-25nt; small interering RNA), further mediation and its homologous single stranded RNA degraded of these little RNA.Based on above-mentioned cognition, the gene constructed one-tenth inverted repeats that is derived from virus is imported plant materials, its transcription product can be by the complementary double-stranded RNA structure that forms of intramolecularly sequence, these double-stranded RNAs further are degraded into little RNA, if donor virus invaded plants body, these little RNA can mediate in the virus mRNA and partly degrade with its homologous, have stoped duplicating and spreading of virus, and the render transgenic plant obtains disease resistance.At present, this respect has some researchs, as (1998) such as Waterhouse the dsRNA of PVY proteinase gene is imported tobacco, makes tobacco obtain special resistance to this virus, and resistance level is higher than just RNA or sense-rna far away; Wang etc. (2000) import barley to the inverted repeats (hpBYDVpol) of barly yellow dwarf virus (Barley yellow dwarf luteovirus-PAV) pol gene, transfer-gen plant is carried out that ELISA detects and aphid is fed and all finds viral, think that resistance level is immune; Smith etc. (2000) import tobacco to the inverted repeats of the proteinase gene of PVY, and disease resistance has also reached immune level; Missiou etc. (2004) import potato to the segmental inverted repeats of the coat protein gene of PVY, have obtained high anti-clone, confirm that disease resistance is RNA interferential result.Usually, people's common practice just imports potato to the segmental inverted repeats of the coat protein gene of PVY, make potato obtain resistance to PVY virus, perhaps just the inverted repeats of the replicase protein gene fragment of PVX is imported potato, make potato obtain the resistance of PVX virus, it all is to utilize an a kind of double stranded rna molecule of artificial genetic expression to make the resistance of plant acquisition to a kind of virus.In order to make plant obtain resistance simultaneously to two kinds of viruses, people also can import the gene fragment of two kinds of viruses in the plant materials respectively, perhaps 1 gene is forwarded in the plant, the transformant that obtains is carried out transgeneic procedure again, the 2nd gene changeed into again, or change individual gene over to recipient plant respectively, hybridize with these 2 kinds of recipient plants again, the filial generation screening is obtained polygenic transformant, perhaps carry out a plurality of carrier cotransformations, but these methods are loaded down with trivial details, time-consuming, and genetic manipulation has repeatedly also brought the difficulty of antibiotic-screening subsequently.When making up aforesaid pair of valency carrier, PVX virus coat protein gene and PVY rdrp virus gene all will have the promotor of oneself, transgenosis will be with 2 promotors in other words, and dicotyledons composition promotor commonly used in fact has only 35S promoter, reuse same promotor in the transgenosis body and cause that easily homology suppresses, and influences genetically modified expression.And utilize RNA interference principle render transgenic plant to obtain resistance to 2 kinds of viruses, or more particularly, the transgenic plant that the double-stranded RNA render transgenic plant that utilizes a genetic expression to go out different sources obtains anti-2 kinds of viruses or multiple virus simultaneously do not appear in the newspapers as yet.
Summary of the invention
The present invention provides a kind of construction process that improves potato to the gene of two resistances of PVX virus and PVY virus in order to solve the existing various problems that improves potato to the method existence of two resistances of two kinds of viruses.
The present invention adopts following technical scheme to realize: improve the construction process of potato to the gene of two resistances of PVX virus and PVY virus, may further comprise the steps: (1) obtains PVX coat protein gene complete sequence cp and PVY rdrp gene complete sequence NIb through extracting total RNA, RT-PCR amplification and gene clone operation respectively in the plant materials that infects PVX virus and PVY virus; (2) utilize pcr amplification to obtain one section conservative gene fragment on PVX coat protein gene complete sequence cp and the PVY rdrp gene complete sequence NIb respectively; (3) enzyme is cut the conservative gene fragment of the PVX coat protein gene complete sequence cp that pcr amplification obtains, couples together to constitute artificial fusion gene cn with one section conservative gene fragment on the PVY rdrp gene complete sequence NIb with identical restriction enzyme site; (4) with artificial fusion gene cn with forward with oppositely be connected respectively to the both sides of an intron, be built into the inverted repeats of an artificial fusion gene, this sequence is and improves the two resistant gene RNAiCNs of potato to X virus and Y virus; (5) the inverted repeats two ends of artificial fusion gene cn are connected with promotor and terminator respectively.
The present invention based on theoretical basis be that a gene constructed one-tenth inverted repeats that is derived from virus is imported a plant materials, its transcription product can be by the complementary double-stranded RNA structure that forms of intramolecularly sequence, these double-stranded RNAs further are degraded into little RNA, if donor virus invaded plants body, these little RNA can mediate in the virus mRNA and partly degrade with its homologous, stoped duplicating and spreading of virus, thereby the render transgenic plant obtains disease resistance.
In the above-mentioned pair of resistant gene RNAiCN building process, the preparation method of PVX coat protein gene complete sequence cp and PVY rdrp gene complete sequence NIb is that those of ordinary skill in the art knows, the method of choosing needed conserved sequence from above-mentioned complete sequence also belongs to prior art, if the restriction endonuclease of selecting for use in above steps is different, the nucleotide sequence of resulting couple of resistant gene RNAiCN is different.
The effect of intron of the present invention is to promote the reverse reiterated DNA sequences of artificial fusion gene to be transcribed into the RNA sequence in plant materials, transcribe the efficient height, speed is fast, and the RNA sequential structure that is transcribed into is stable, and the intravital intron of any plant of the present invention can be used.
The effect of promotor of the present invention is to start to transcribe, and selects 35S usually for use, and terminator is selected NOS poly-A usually for use, and this is that those of ordinary skill in the art knows.
Two resistant gene RNAiCN that the present invention is built by the DNA recombinant technology be connected to can the plant expression vector of self-replacation in bacterial cell or vegetable cell on, for example be derived from colibacillary plasmid vector pUC18, pUC19, and the plasmid vector (producing) and the pBS-T carrier (producing) that cause a series of pGEM of being commonly referred to as of different multiple clone site through different modifications by sky root company by Promega company, especially plant expression vector pCAMBIA1301, pBin19, pROKII and the (Jefferson of pBI131 system, et al., EMBO J, 16:3901,1987) etc., in a series of preferred embodiments of the present invention, select pUC19 for use, pCAMBIA1301 etc., the latter are particularly suitable for the instrument plasmid vector as the preparation plant expression vector; Plant expression vector is changed over to and is incorporated in the potato gene group then, and the expression of two resistant gene RNAiCN in potato can be given the two resistances of this plant to PVX and two kinds of viruses of PVY, and offspring and the organizing of any part of potato all have two resistances simultaneously.Above-mentionedly will carry that the expression of exogenous gene carrier imports host plant or its intracellular many methods all are well known to those skilled in the art.These methods comprise a, agrobacterium-mediated transformation, b, physics method, as particle bombardment, electric shock fusion method, microinjection, supersonic method etc.; C, chemical method are as the conversion method of PEG mediation, liposome-mediated conversion method etc.; D, implant system conversion method are as pollen tube passage method, ovary injection, infusion method etc.; E, with conversion methods that virus vector was mediated such as cauliflower mosaic virus (CaMV), geminivirus infection (Geminiviruses) or RNA viruses.
Compared with prior art, the present invention has the following advantages:
(1) can suppress duplicating of two kinds of viruses simultaneously, the render transgenic potato obtains the two resistances to PVX virus and PVY virus simultaneously, effectively solves the shrinkage flower leaf type that exists in the potato production and degenerates;
(2) transcription product of the fusion gene inverted repeats of method structure of the present invention can form a kind of sequence oneself complementary double-stranded RNA structure, this structure can excite the intravital RNAi mechanism of plant, this duplex structure is degraded into small segment, therefore genetically modified mRNA do not exist or amount seldom, can not translate into protein yet, avoid conventional antiviral transgenosis mode may cause the potential source biomolecule risk that virus and transgenosis generation homologous recombination or allos are packed;
(3) with fusion gene of two gene constructed one-tenth, two genes get final product with a promotor, have also just avoided suppressing problem by the homology of using identical promoters to cause, simultaneously, utilize a carrier, once transform and get final product, and are time saving and energy saving;
In a word, after the fusion gene inverted repeats that utilizes method of the present invention to make up imported potato, it was to the two anti-strain rate height of two kinds of viruses, and resistance can reach high anti-, even immune level, more easily application on producing.
Description of drawings
Fig. 1 is the building process synoptic diagram of of the present invention couple of resistant gene RNAiCN
Fig. 2 cuts qualification result for the enzyme to the plant expression vector pRNAiCN of two resistant gene RNAiCN in the embodiment of the invention
Fig. 3 is to many primer PCRs amplification qualification results of the plant expression vector pRNAiCN of two resistant gene RNAiCN in the embodiment of the invention
Embodiment
Below be a specific embodiment of the present invention:
Step 1: the acquisition of PVX coat protein gene complete sequence cp and PVY rdrp gene complete sequence NIb
1, the acquisition of PVX coat protein gene complete sequence cp
1) according to the nucleotide sequence (accession:AF528555) of PVX coat protein gene complete sequence cp, design the PCR primer:
Forward primer: 5 '-ATGTCAGCACCAGCTAGCAC-3 ';
Reverse primer: 5 '-TTATGGTGGTGGTAGAGTGA-3 '.
Primer is synthetic by three rich companies.
2) full length DNA sequence article one chain of PVX coat protein gene cp is synthetic: extract total RNA of the tobacco leaf that infects PVX virus, get 2 μ L, add the reverse primer 2 μ L of 10 μ M again, 10mM dNTP 2 μ L, ddH2O 12 μ L.65 ℃ of water-bath 5min, ice bath 2min.In pipe, add following component again: 5 * RT damping fluid, 5 μ L, RNasin1 μ L (5u), M-MLVRT enzyme 1 μ L (100u) behind the mixing, reacts by following condition: elder generation 42 ℃ of 60min, 85 ℃ of 10min termination reactions again.
3) pcr amplification: with step 2) the reverse transcription synthetic first chain cDNA is a template, under the guiding of forward primer and reverse primer, and the full-length cDNA gene of pcr amplification PVY rdrp virus.After the PCR reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, reclaim the amplified production that test kit (sky is the epoch) reclaims about 720bp with pillar.
4) gene clone: get the PCR product that step 3) reclaims and is connected with pGEM-T Easy carrier, transformed into escherichia coli DH5 α competent cell, the upgrading grain carries out enzyme and cuts evaluation then, send three to win with Bo Ya company and carry out nucleotide sequencing positive colony.Sequencing result shows that the full-length cDNA of PVX coat protein has inserted pGEM-T Easy carrier, and the recon that obtains is called after pGEM-PVXCP respectively.
2, the acquisition of PVY rdrp gene complete sequence NIb
1) nucleotide sequence (accession:NC001616) according to PVY rdrp virus gene NIb designs the PCR primer:
Forward primer: 5 '-GCTAAGCATTCTGCATGGATG-3 ';
Reverse primer: 5 '-TGCATCAATTGTGTCATTTGC-3 '.
Primer is synthetic by three rich companies.
2) PVY rdrp gene NIb full-length cDNA article one chain is synthetic: extract total RNA of the tobacco leaf that infects PVY virus, get 2 μ L, add the reverse primer 2 μ L of 10 μ M again, 10mM dNTP 2 μ L, ddH
2O 12 μ L.65 ℃ of water-bath 5min, ice bath 2min.In pipe, add following component again: 5 * RT damping fluid, 5 μ L, RNasin1 μ L (5u), M-MLVRT enzyme 1 μ L (100u) behind the mixing, reacts by following condition: elder generation 42 ℃ of 60min, 85 ℃ of 10min termination reactions again.
3) pcr amplification: with step 2) the reverse transcription synthetic first chain cDNA is a template, under the guiding of forward primer and reverse primer, and the full-length cDNA gene of pcr amplification PVY rdrp virus.After the PCR reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis detect, reclaim the amplified production that test kit (sky is the epoch) reclaims about 1600bp with pillar.
4) gene clone: get the PCR product that step 3) reclaims and is connected with pGEM-T Easy carrier, transformed into escherichia coli DH5 α competent cell, the upgrading grain carries out enzyme and cuts evaluation then, send three to win with Bo Ya company and carry out nucleotide sequencing positive colony.Sequencing result shows that the full-length cDNA of PVY replicative enzyme NIb has inserted pGEM-T Easy carrier, and the recon that obtains is called after pGEM-PVYNIb respectively.
Step 2: the structure of two resistant gene RNAiCN and plant expression vector thereof
1, the structure of fusion gene cn
1) utilize BLAST software with the PVX coat protein gene complete sequence cp of being cloned into and the sequence alignment among the GeneBank, the fragment of choosing the higher 517nt of conservative property from complete sequence is used to make up the RNAi gene.This 517nt is positioned at 126-642 the Nucleotide place of PVX coat protein gene complete sequence cp.
2) with BLAST software with the PVY rdrp gene complete sequence NIb of being cloned into and the sequence alignment among the GeneBank, the fragment of choosing the higher 546nt of conservative property from complete sequence is used to make up the RNAi carrier.The fragment of this 546nt is positioned at 587-1132 the Nucleotide place of PVY rdrp gene complete sequence NIb.
3) with pGEM-PVXCP be template, 5 '-CTG GGT ACC AGT AGC CAG CAA TGC CG-3 ') and 5 ' hold that the people is arranged is that the reverse primer (5 '-CGG TCT AGA GTG ACA GCT GCA TCT AG-3 ') that adds the XbaI enzyme cutting site carries out pcr amplification with 5 ' end the people being arranged is that (primer sequence:, the PCR product is the dna fragmentation of the 517nt of the two ends coat protein gene cp that has KpnI and XbaI enzyme cutting site respectively for the forward primer that adds the KpnI restriction enzyme site.
4) with KpnI and XbaI double digestion PCR product, with the directed upstream of inserting the NIb gene fragment of the 546nt that relatively guards on the carrier pGEM-PVYNIb of endonuclease bamhi, be built into the fusion gene cn that forms by PVX cp gene fragment and PVY NIb gene fragment, the recombinant vectors called after pGEM-CN of structure;
5) with pGEM-PVYNIb be template, 5 '-CAC TCG AGA TAC CTT GCT GGG TGG T-3 ') and 5 ' hold that the people is arranged is that (primer sequence: 5 '-CGG TTA CCA AGA TCT GAG AAGTGT-3 ') carry out pcr amplification, the PCR product is the dna fragmentation of the 546nt of the two ends rdrp gene NIb that has XhoI and BstEII restriction enzyme site respectively for the reverse primer that adds the BstEII restriction enzyme site with 5 ' end the people being arranged is the forward primer that adds the XhoI restriction enzyme site (primer sequence:.
This PCR product is connected with the pBS-T carrier, makes up recombinant vectors pBS-PVYmNIb.
6) with pGEM-PVXCP be template, 5 '-CTG ATC GAT AGC CAG CAA TGC CG-3 ') and 5 ' hold that the people is arranged is the reverse primer that adds the XhoI restriction enzyme site (primer sequence: 5 '-CGG CTC GAG GTG ACA GCT GCA TCT AG-3 ') carry out pcr amplification, obtain the dna fragmentation of 517nt that two ends have the coat protein gene cp of ClaI and XhoI restriction enzyme site respectively with 5 ' end the people being arranged is the forward primer that adds the ClaI restriction enzyme site (primer sequence:;
With ClaI and XhoI double digestion PCR product,, be built into the 2nd fusion gene cn, recombinant vectors called after pBS-CN with the directed upstream of inserting NIb gene fragment on the pBS-PVYmNIb carrier of endonuclease bamhi.
2, the structure of two resistant gene RNAiCN and expression vector thereof
1) with ClaI and KpnI double digestion carrier pKANNIBAL, on the directed pGEM-CN of insertion of intron pdk that downcuts, cn oppositely links to each other with fusion gene, forms fusion gene-intron multiplexed sequence cn-pdk, recombinant vectors called after pGEM-CNP;
2) with isocaudarner XhoI and SalI double digestion plant expression vector pCAMBIA-1301, excise the expression cassette of the marker gene npt II on the pCAMBIA-1301, obtain the pCAMBIA-1301 of marker-free;
3) with BglII and ClaI double digestion recombinant vectors pGEM-CNP, downcut fusion gene-intron multiplexed sequence cn-pdk;
4) with BstEII and ClaI double digestion recombinant vectors pBS-CN, downcut fusion gene cn;
5) fusion gene-intron multiplexed sequence cn-pdk and fusion gene cn are inserted simultaneously BglII and the BstEII incision of the pCAMBIA-1301 of marker-free, make fusion gene cn respectively with forward and the both sides that are reversely connected to intron pdk, be built into the inverted repeats of an artificial fusion gene, this sequence is and improves the two resistant gene RNAiCNs of potato to X virus and Y virus.The recombinant vectors that obtains is exactly the plant expression vector of two resistant gene RNAiCN, called after pRNAiCN.Its plasmid map and building process thereof are seen accompanying drawing 1.
6) the plant expression vector pRNAiCN of RNAiCN gene being carried out multienzyme cuts and identifies and many primer PCRs amplification evaluation, enzyme is cut qualification result and is seen that (swimming lane 1 is HindIII+ClaI double digestion product to accompanying drawing 2, swimming lane 2 is a HindIII+KpnI double digestion product, swimming lane 3 is a BstEII+XbaI double digestion product, swimming lane 4 is an XbaI single endonuclease digestion product, swimming lane 5 is a PstI single endonuclease digestion product, swimming lane 6 is a BglII single endonuclease digestion product), many primer PCR amplification qualification results see that (swimming lane 1 is the amplified production of primer P1 and P2 to accompanying drawing 3; Swimming lane 2 is the amplified production of primer P3 and P4; Swimming lane 3 is the amplified production of primer P4 and P5; Swimming lane 4 is the amplified production of primer P4 and P6; Swimming lane 5 is the amplified production of primer P5 and P7), it is correct to show that pRNAiCN makes up.
The primer sequence that pcr amplification is identified is:
P1：5’-GTGTAAGACGAAGAAGAAGAT-3’；
P2：5’-GATCTATCATGTTACCTTG-3’；
P3：5’-AGATACCTTGCTGGGTGGT-3’；
P4：5’-AACCAAGATCTGAGAAGTG-3’；
P5：5’-ATAGTAGCCAGCAATGCCG-3’；
P6：5’-TACCTGAGAATTGGGTATAC-3’；
P7：5’-GAGTGACAGCTGCATCTAG-3’.
With agrobacterium-mediated transformation the plant expression vector pRNAiCN of two resistant gene RNAiCN of making up in the step 3 is imported potato, concrete grammar may further comprise the steps:
1, preparation Agrobacterium LBA4404 competent cell
Get 28 ℃ of shaking culture to OD
600The bacterium liquid that is 0.5 Agrobacterium LBA4404 is abandoned supernatant behind the centrifugal 5min of 5000rpm, add the NaCl solution suspension cell of 10mL 0.15M then, abandons supernatant behind the centrifugal 5min of 5000rpm again, with the 20mM CaCl of 1mL precooling
2Suspension cell, packing behind the ice bath, quick-frozen is 1 minute in the liquid nitrogen, and it is standby to put-70 ℃ of preservations;
2, by the direct conversion method of freeze thawing the fusion gene inverted repeats is imported Agrobacterium LBA4404
Get the competent cell of the Agrobacterium LBA4404 of 200 μ L steps 1 preparation, the plant expression vector pRNAiCN that adds the RNAi gene of 1 μ g embodiment, 2 structures, quick-frozen 1min in the liquid nitrogen, 37 ℃ of water-bath 5min, add 1mL YEB substratum then, 28 ℃ at a slow speed behind the shaking culture 4h in the centrifugal 30sec of 1000rpm, abandon supernatant, add 0.1mL YEB substratum suspension cell again, coat on the YEB flat board that contains 100 μ g/mL kantlex (Kan) and 125 μ g/mL Streptomycin sulphates (Sm), behind 28 ℃ of cultivation 48h, the single bacterium colony that grows on the picking flat board, be inoculated in the YEB liquid nutrient medium (containing 100 μ g/mL kantlex and 125 μ g/mL Sm), 28 ℃ of shaking culture are spent the night.Bacterium liquid is carried out PCR identify, obtain the Agrobacterium of recombinating, called after LBA4404/pRNAiCN.
Carry out PCR and identify that the primer sequence is:
PrimeF：5’-CATGAAGGTGCCCACAGAC-3’；
PrimeR：5’-CGGATTCACAGCAATCAGC-3’。
3, by the RNAi gene importing potato of agrobacterium-mediated transformation with plant expression vector.
The single bacterium colony of picking Agrobacterium LBA4404/pRNAiCN is in 5ml YEB liquid nutrient medium (containing 100mg/LSm, 100mg/L Kan), and 28 ℃, 150rpm shaking culture 16-20h was transferred in the YEB liquid nutrient medium according to 1: 100, and shaking culture is to OD
6000.5; The centrifugal 10min of 6000rpm abandons supernatant.With the centrifugal gained precipitation 12ml 10mM MgSO of 30ml bacterium liquid
4Again suspend, the centrifugal 10min of 6000rpm abandons supernatant.(MS minimum medium+1.6% glucose+5mg/l NAA+0.1mg/l6-BA pH5.3) suspends again, repeats once to remove microbiotic again, and dilution bacterium liquid is to OD with high sugar liquors substratum
600Be 0.5, diluent is poured in the sterilization culture dish.The blade of cultivating the potato aseptic seedling on the MS minimum medium is cut along leaf base, vertical master pulse crosscut 2-3 cutter (every long incision 0.1-1cm, between otch at a distance of 4-5mm), the leaf face down floats in the bacterium liquid of dilution, after shaking mixing gently, leave standstill 30min, after change high sugared solid medium (pH5.8) over to and cultivate 4d altogether.Then explant is put into division culture medium (MS minimum medium+0.02mg/l NAA+0.02mg GA
3+ 2.0mg/Zeatin+250mg/l Cef) illumination cultivation is induced and is sprouted, and every 20d subculture 1 time when treating that bud grows to 1-1.5cm, changes it in MS minimum medium root induction.
4, the Molecular Detection of transgenic Rhizoma Solani tuber osi
Cut a small amount of blade from the potato test-tube plantlet of differentiation, extract genomic dna with the SDS method, get 100ng, carry out pcr amplification, the while is made positive control with the amplified production of the carrier pRNAiCN of structure; Amplified production with the genomic dna that extracts with the SDS method of kind non-transgenic potato plant is made negative control.Reaction conditions is: 94 ℃ of sex change 5min; 94 ℃ of sex change 1min; 56 ℃ of renaturation 1min, 72 ℃ are extended 1min30s, circulate 30 times; Mend flat 8min for 72 ℃.The PCR product is detected with 1% agarose gel electrophoresis.The result shows has 18 strains can amplify the specific band of 949bp, and positive transfer-gen plant, negative control do not amplify this specific band.
Pcr amplification the primer sequence is:
PrimeF：5’-CATGAAGGTGCCCACAGAC-3’；
PrimeR：5’-CGGATTCACAGCAATCAGC-3’。
PCR is detected positive, the normal transfer-gen plant of growing, picked at random 8 strains and 1 strain non-transgenic potato adopt the SDS method to extract plant genome DNA, get 20 μ g, with the HindIII enzyme cut spend the night after, agarose gel electrophoresis with 0.8%, transfer on the positively charged nylon membrane with the capillary transfer method afterwards, fusion fragment with digoxigenin labeled is a probe, carry out Southern hybridization by test kit explanation (DIG High Prime Labeling andDetection Starter Kit I), the result shows that 8 strain transfer-gen plants all have These positive bands to occur, but not transfer-gen plant does not then have corresponding These positive bands to occur.
To be decided to be transfer-gen plant through PCR detection and 8 positive plant of Southern hybridization.These 8 transfer-gen plants are carried out vegetative propagation, form 8 transgenosis clones.Each clone is transplanted 3 strains in the greenhouse, strain inoculation PVX, and strain inoculation PVY, PVX and PVY are inoculated in another strain simultaneously, to compare with kind non-transgenic potato, observe disease symptom simultaneously, and make DAS-ELISA and analyze.
The inoculation method of PVX and PVY virus is: the blade that will infect PVX and PVY virus respectively is ground to Powdered in liquid nitrogen, and (filling a prescription is Na for 0.02M PB, pH7.0 to add the extraction damping fluid of 10 times of volumes (W/V)
2HPO
4.12H
2O, 43.7g, NaH
2PO
4.H
2O 12.2g, distilled water is settled to 1L), shake mixing after the centrifugal 5min of 4000rpm get supernatant.Treat the potato test-tube plantlet transplant survival after 1 month, sprinkle 600 order silicon carbide the middle and upper part face of blade, cotton balls dip in viral juice after unidirectional twice, 1 week of friction again repeated inoculation virus once inoculate altogether 3 times.
With the PVX-IgG of alkali phosphatase enzyme mark and PVY-IgG as antibody.Get 0.5g blade liquid nitrogen grinding for the every strain of examination potato, add 5ml sample extraction damping fluid (137mM NaCl, 1.5mM KH
2PO
4, 8.1mMKH
2PO
4, 2.7mM KCl and 3.1mM NaCl
2, Ph7.4, and additional 0.2% skim-milk, 0.05%Tween20,2%PVP (M44000) homogenate), with PVX in double-antibody sandwich enzyme linked immunological absorption (DAS-ELISA) method mensuration transgenic potato plant and the content of PVY.On every elisa plate, be equipped with the transfer-gen plant not negative control and the positive control of transfer-gen plant virus inoculation not of virus inoculation.The colour developing back joins the instrument reading with BIO-RAD 550 enzymes, determines the OD of each plant
405Value.Each sample is all established 3 repetitions.
To gained 8 potato transgenosiss clone and 1 PVX and PVY inoculation test that non-transgenic potato clone is carried out, observation of symptoms result shows: 8 transgenic Rhizoma Solani tuber osi clones are all to PVX and PVY performance immunity, flower leaf paresthesia does not appear in blade, non-transgenic potato clone is all very sensitive to PVX and PVY, and blade table reveals flower leaf paresthesia.The ELISA value of surveying also consistent with the observation of symptoms result.
Claims (1)
1. a construction process that improves potato to two resistant genes of X virus and Y virus is characterized in that may further comprise the steps,
(1) in the plant materials that infects PVX virus and PVY virus, obtains PVX coat protein gene complete sequence cp and PVY rdrp gene complete sequence NIb respectively through extracting total RNA, RT-PCR amplification and gene clone operation;
(2) utilize pcr amplification to obtain one section conservative gene fragment on PVX coat protein gene complete sequence cp and the PVY rdrp gene complete sequence NIb respectively, the conservative gene fragment is positioned at 126-642 the Nucleotide place of PVX coat protein gene complete sequence cp and 587-1132 the Nucleotide place of PVY rdrp gene complete sequence NIb;
(3) enzyme is cut the conservative gene fragment of the PVX coat protein gene complete sequence cp that pcr amplification obtains, couples together to constitute artificial fusion gene cn with one section conservative gene fragment on the PVY rdrp gene complete sequence NIb with identical restriction enzyme site;
(4) with artificial fusion gene cn with forward with oppositely be connected respectively to the both sides of pdk intron, be built into the inverted repeats of an artificial fusion gene, this sequence is and improves the two resistant gene RNAiCNs of potato to X virus and Y virus;
(5) the inverted repeats two ends of artificial fusion gene cn are connected with promotor and terminator respectively.
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| US4970168A (en) * | 1989-01-27 | 1990-11-13 | Monsanto Company | Virus-resistant plants |
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| US4970168A (en) * | 1989-01-27 | 1990-11-13 | Monsanto Company | Virus-resistant plants |
| US6015940A (en) * | 1992-04-07 | 2000-01-18 | Monsanto Company | Virus resistant potato plants |
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| Title |
|---|
| JP平1-281079 1989.11.13 |
| 丁红梅等.内含子与基因表达调控.畜牧与兽医38 3.2006,38(3),50-52. |
| 丁红梅等.内含子与基因表达调控.畜牧与兽医38 3.2006,38(3),50-52. * |
| 白庆荣.利用RNA介导的病毒抗性培育双抗病毒转基因植株.山东农业大学 博士学位论文.2004,参见摘要部分第2段、第3段、第2.2节、第2.4节. * |
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