CN107090469B - A method for reducing seed shattering of hybrid offspring of cultivated rice and weedy rice by using SH4 gene silencing - Google Patents
A method for reducing seed shattering of hybrid offspring of cultivated rice and weedy rice by using SH4 gene silencing Download PDFInfo
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Abstract
本发明属于生物环境安全检测技术领域，具体为一种利用SH4基因沉默降低栽培稻与杂草稻杂交后代种子落粒性的方法。本发明通过基因沉默技术使栽培稻的落粒基因（SH4）功能丧失，含SH4基因沉默元件的栽培稻转基因一旦通过基因漂移转移到杂草稻群体，由于种子落粒性下降，将降低转基因扩散的风险；同时，包含该SH4基因沉默元件的杂草稻群体的种子落粒性和扩散能力将大大下降，从而可以通过栽培稻向杂草稻的基因漂移，将该落粒性降低的性状转移到杂草稻群体，达到控制杂草稻群体扩散，降低杂草稻危害的目的。本发明能够极大降低转基因逃逸到杂草稻之后可能导致的环境安全风险，降低杂草稻扩散和危害，避免经济损失，具有广阔应用前景。
The invention belongs to the technical field of biological environment safety detection, in particular to a method for reducing seed shattering of hybrid offspring of cultivated rice and weedy rice by utilizing SH4 gene silencing. In the present invention, the function of the shattering gene ( SH4 ) of cultivated rice is lost through the gene silencing technology. Once the cultivated rice transgene containing the SH4 gene silencing element is transferred to the weedy rice population through gene drift, the seed shattering property is reduced, and the spread of the transgene will be reduced. At the same time, the seed shattering and spreading ability of weedy rice populations containing the SH4 gene silencing element will be greatly reduced, so that the trait of reduced seed shattering can be transferred through gene drift from cultivated rice to weedy rice To the weedy rice population, to control the spread of the weedy rice population and reduce the harm of the weedy rice. The invention can greatly reduce the environmental safety risk that may be caused after the transgene escapes to the weedy rice, reduce the spread and harm of the weedy rice, avoid economic losses, and has broad application prospects.
Description
Technical Field
The invention belongs to the technical field of biological environment safety detection, and particularly relates to a method for reducing seed shattering of hybrid progeny seeds of cultivated rice and weedy rice.
Background
The commercial application of transgenic crops brings great economic benefits, and simultaneously, potential transgenic organism safety problems exist: some transgenes (genes of interest) with natural selection advantages, such as: disease, insect and herbicide resistant transgenes can escape from cultivated crops to their wild kindred species by gene drift, thereby generating weeds which are difficult to control and bringing about potential environmental biosafety problems. Thus, in order to try to avoid or reduce the possible hazards due to transgene escape, techniques can be attenuated by transgenes, namely: the gene which has inferior expression on wild relative species of the cultivated crops but has no influence or is beneficial to the cultivated crops is transferred into the cultivated crops, and the gene is transferred into the wild relative population of the cultivated crops through gene drift, thereby achieving the purpose of reducing (weakening) the suitability of the wild relative species. The weakening gene is used as a safety box to be connected with a target transgene in series to construct a transgene vector, and the transgene vector is transferred into a cultivated crop together, so that the environmental safety risk possibly brought by the escape of the target transgene to wild kindred species of the cultivated crop can be reduced.
Cultivated rice is one of the most important grain crops in the world. In recent years, the number of weedy rice in paddy fields has increased due to various reasons such as changes in the way of growing cultivated rice. The weedy rice has stronger seed falling property, and mature rice seeds can easily fall into soil of a rice field under the action of natural wind power or external mechanical force and can not be harvested, so that grains are harvestedA huge loss of yield. However, cultivated rice has poor seed shattering properties. The study showed that the shattering gene of cultivated rice (SH4) Is one of key genes causing the reduction of seed shattering performance in the domestication process, and influences the seed shattering performance of the cultivated rice by regulating and controlling the formation and degradation of a delamination layer on a small spike-cob of the mature cultivated rice. Inhibition can be achieved by different methods of gene silencingSH4Expression of the gene, thereby reducing seed shattering of cultivated rice ifSH4Entry of the gene silencing element into the weedy rice population by gene drift reduces seed shattering of the weedy rice population. Therefore, there is an urgent need for a method of cultivating rice using silent rice plantsSH4The gene weakens the falling performance of the weedy rice seeds by the drift of the gene from the cultivated rice to the weedy rice, thereby reducing the environmental safety risk possibly brought by the transfer of the transgenes from the transgenic cultivated rice to the weedy rice, and simultaneously reducing the harm of the weedy rice in the rice field by reducing the falling performance of the weedy rice.
Disclosure of Invention
The purpose of the invention is to provide a method for utilizingSH4A gene silencing technology and a method for reducing the seed shattering property of cultivated rice and hybrid progeny of the cultivated rice and weedy rice.
The invention is prepared by synthesisSH4The gene silencing element is fused with a promoter capable of efficiently expressing in a rice seedling abscission layer tissue to form a chimeric gene, and a plant expression vector containing the chimeric gene is introduced into rice seedlings through genetic transformation to obtain 'safe' transgenic rice seedlings capable of reducing the risk of transgene escape. The transgenic cultivated rice can reduce the expression of plant on the carrierSH4Any transgenes tightly connected with the gene silencing element can be diffused in the weedy rice group and continuously cause the possibility of harm, so that the environmental biosafety of the transgenic cultivated rice is improved, and meanwhile, the diffusion of the weedy rice can be prevented through the technology. The method has wide application value and popularization prospect in the aspects of transgenic crop breeding and commercial production.
In particular, the invention provides the use ofSH4The gene silencing element is used in reducing seed falling performance of rice and hybrid rice and weedy riceThe rice shattering gene in cultivated rice due to silencing technique (SH4) Loss of function when silentSH4The gene is introgressed into the weedy rice group through hybridization, so that the seed shattering property of the hybrid progeny group of the cultivated rice and the weedy rice can be reduced, and the technical method for reducing the seed shattering property of the hybrid group of the transgenic cultivated rice and the weedy rice is established. The method comprises the following specific steps:
(1) synthesis ofSH4A gene silencing element;
(2) will be able to driveSH4Plant expression promoter with gene silencing element for high-efficiency expression in rice seedling abscission layer tissue, such as Ubi promoter or pSH4 promoter, and the likeSH4Fusing the gene silencing elements to form a chimeric gene;
(3) introducing the chimeric gene into cultivated rice by a transgenic method by using a plant expression vector such as pCXUN, pCXUN-osaMIR528, pCAMBIA1301, pTCK303 or the like to obtain a transgenic cultivated rice line;
(4) simulating the process of the cultivated rice gene drifting to the weedy rice, and artificially hybridizing the transgenic cultivated rice line and the weedy rice to obtain a hybrid F1Generation group, hybrid F1The generation group is bred by self-crossing to obtain a hybrid F2And (4) generation groups. By pairing hybrids F1Generation population and hybrid F2The generation group is subjected to transgenic component identification, and can be classified as containingSH4Transgenic populations of gene silencing elements (F)1+，F2+) And a population without transgenes (F)1-，F2-). For hybridization F1Generation population and hybrid F2The generation group is planted in the field, the seed falling property is measured and calculated in the plant mature period (SHI). When it containsSH4Of transgenic populations of gene silencing elementsSHIValue ratio of the population without transgeneSHIA low value indicatesSH4The gene silencing element reduces the seed falling property of filial generation of the cultivated rice and the weedy rice, thereby establishing and utilizingSH4A method for reducing seed shattering of filial generation of oryza sativa and oryza sativa by using a gene silencing element.
In the present invention, the step (1) is synthesizedSH4Gene silencing elements are of two types, namelyIn addition, the gene silencing element is an artificial microRNA gene silencing element, and the RNA interference gene silencing element has a hairpin structure. Both silencing elements have the same effect.
(1) The artificial microRNA gene silencing element comprises the following specific synthetic steps: cultivating rice containing and encodingSH4Replacing endogenous gene of Oryza sativa with a nucleotide sequence of which the difference between consecutive 21 nucleotide sequences in the cDNA nucleotide sequence of the gene is not more than 2 bases and the nucleotide sequence complementary theretoosaMIR528The corresponding gene silencing nucleotide sequence of (a), capable of forming a double-stranded RNA molecule in a plant;
the specific method for synthesizing the artificial microRNA gene silencing element comprises the following steps: according to the technical principle of artificial microRNA gene silencing, rice shattering genes are cultivated on an online tool platform (http:// WMD3. weightelworld. org) of Web MicroRNA Designer (WMD)SH4The genome sequence (LOC _ Os04g57530.1, TIGR oryza sativa whole genome annotation database) is taken as a target sequence, and screening can be carried out to realize silenceSH4Artificial microRNA fragments with most suitable genes. Then, according to the overlapping PCR steps (Table 1), the endogenous microRNA precursor osa-MIR528 of the cultivated rice is used as a template (the template is from a pNW55 vector), an artificial microRNA specific primer pair is designed by a WMD online tool (Table 2), the artificial microRNA fragments are respectively replaced by corresponding gene specific silent fragments in the osa-MIR528 precursor, and the specific silent fragments are synthesizedSH4Artificial microRNA silencing elements of genes. The steps of overlapping PCR included 4 PCR reactions (Table 1).
TABLE 1 overlapping PCR steps for construction of artificial microRNA gene silencing elements
The proper artificial microRNA fragment preferably has both specificity and high efficiency, and the specificity ensures that the artificial microRNA fragment does not take other genes in a genome as targets to cause non-specific gene silencing, namely 'off-target'; the efficient artificial microRNA fragment has 21 bases, wherein the 2 nd to 12 th bases are completely complementary with a target sequence and have no mismatch, and the 18 th to 21 th bases at the 3' end of the efficient artificial microRNA fragment preferably have 1 to 2 mismatches. Furthermore, the absolute hybridization energy between the artificial microRNA fragment and the target sequence should be less than-30 kcal/mole, preferably between-35 and-40 kcal/mole.
Table 2 overlapping PCR primers for constructing artificial microRNA gene silencing element
Note: underlined nucleotide bases of the primers indicate alternative specific silent oryza sativaSH4Artificial microRNA fragments of genes. The primers are sequentially marked as SEQ.ID.NO.1, SEQ.ID.NO.2, SEQ.ID.NO.3, SEQ.ID.NO.4, SEQ.ID.NO.5 and SEQ.ID.NO. 6.
The PCR reaction system and the reaction program of each reaction step for synthesizing the artificial microRNA fragment are as follows:
the PCR reaction system of reaction steps a-c:
PCR reaction procedure:
and c, after the PCR reaction in each step a-c is finished, carrying out 2% (v/w) agarose gel electrophoresis on the obtained product, tapping, recovering, and separating and purifying the corresponding target fragment. And dissolving the recovered a-c fragment in water to be used as a template for the final step d of the reaction for synthesizing the artificial microRNA fragment.
And d, a PCR reaction system of the reaction step d:
the PCR reaction procedure was as follows:
the PCR reaction product of reaction step d was isolated and purified on a 1% (v/w) agarose gel. Finally obtaining specific silenceSH4Artificial microRNA gene silencing element of gene.
(2) The RNA interference gene silencing element with hairpin structure is synthesized through the following steps: cultivated rice containing and encodingSH4The cDNA nucleotide sequence of the gene has about 100 to 300 continuous nucleotide sequences with at least 90 percent of sequence similarity and the nucleotide sequence complementary with the continuous nucleotide sequences, and is separated by an intron in a plant expression vector pTCK303, and can form a double-stranded RNA molecule in a plant body.
According to the design principle of RNA interference with hairpin structure, the cultivated rice isSH4The full-length sequence of the gene cDNA was segmented into consecutive short nucleotide fragments of about 100-300 base pairs in length and these short fragments were aligned for online BLAST sequence homology on the NCBI website (http:// www.ncbi.nlm.nih.gov /) with the sequence database of the known Expressed Sequence Tag (EST) from cultivated rice. In principle, as long as there is no match of the continuous 21 bases with the expression sequence of other genes in the genome of Oryza sativa, it is considered that the nucleotide sequence does not pose a risk of nonspecific gene silencing when it functions as RNA interference, and thus can be used as a candidateSH4A gene silencing element. To be provided withSH4Synthesis of specific silencing exemplified by the 5' untranslated region of the cDNA sequence of the gene and a continuous nucleotide fragment (sh 4 ri-3) of 163 base pairs in length from (-137 to 26) in the first exonSH4The synthesis process of RNA interference gene silencing element with hairpin structure of gene is as follows:
a pair of primers for synthesizing sh4ri-3 nucleotide sequence was designed:
sh4ri3-ma：5’-GGTACCACTAGTCCGTGCGGCCTTGTAAA（SEQ.ID.NO.7）
sh4ri3-mb：5’-GGATCCGAGCTCAGCCGTTCTTCCAGCAGTAG，（SEQ.ID.NO.8）。
SpeI and KpnI cleavage sites (underlined bases in sh4ri 3-ma) and SacI and BamHI cleavage sites (underlined bases in sh4ri 3-mb) were added to both sides of the primer pair in order to facilitate ligation of the RNA interference fragment to a specific cloning site in the pTCK303 vector to form a hairpin structure, respectively. The RNA interference fragment is then synthesized by PCR,
the PCR reaction system and reaction procedure were as follows:
and (3) PCR reaction system:
PCR reaction procedure:
the PCR reaction product was separated and purified on 1% (v/w) agarose gel to obtain a DNA fragment againstSH4RNA of the gene interferes with gene silencing fragments. Subsequently, the RNA interference gene silencing fragment is subjected to SpeI/SacI double enzyme digestion and is connected with a plant expression vector pTCK303 which is also subjected to SpeI/SacI double enzyme digestion to form an intermediate vector pTCK 303-a. Then, the same RNA interference gene silencing fragment is subjected to KpnI/BamHI double enzyme digestion and is connected with an intermediate vector pTCK303-a which is also subjected to KpnI/BamHI double enzyme digestion. Finally obtaining the specific silencingSH4RNA with hairpin structure of gene interferes with gene silencing element. The specific steps of the enzyme digestion and ligation reaction are as follows:
enzyme digestion reaction: preparing a vector plasmid to be subjected to (double) enzyme digestion or a PCR product into an enzyme digestion reaction system according to the following system:
the vector plasmid or the PCR product is subsequently cleaved at the optimum temperature for the restriction enzyme used (typically 37 ℃) for about 12 hours. After completion of the cleavage reaction, the resulting product was separated on a 1% (v/w) agarose gel and the desired fragment was recovered for ligation.
Connecting reaction: the vector plasmid large fragment obtained by the enzyme digestion reaction and the small fragment or PCR product are mixed according to a certain proportion by using a ligation kit to carry out ligation reaction for about 12 hours under the condition of 16 ℃ water bath, and the recombinant plant expression vector plasmid can be obtained.
Connecting a reaction system:
in the present invention, the plant expression promoter in step (2) is capable of activatingSH4The gene silencing element is expressed efficiently in the rice seedling abscission layer tissue and comprises a constitutive strong expression promoter or a abscission layer tissue specific expression promoter. Wherein the constitutive strong expression promoter is selected from the promoter Ubi from the ubiquitin gene of Zea mays, and the promoter for the specific expression of the delamination tissue is selected from the shatter gene of oryza sativaSH4The promoter of (3), pSH 4.
In the invention, the specific process of the step (3) is as follows: (1) agrobacterium transformation, culture and identification; (2) genetic transformation of cultivated rice embryogenic calli.
(1) Agrobacterium transformation, culture and identification
Mixing cultivated riceSH4The recombinant plant expression vector plasmid of the gene silencing element is transferred into agrobacterium (A) by an electric shock transformation methodAgrobacteriumtumefaciens) Strain EHA 105. After the Agrobacterium was electrically shocked to absorb the plasmid, 1000. mu.l of YEB medium (Agrobacterium rhizogenes medium) containing no antibiotic was added, the mixture was shaken in a shaker at 28 ℃ for 2 hours (100 rpm), centrifuged briefly, the cell was collected and the majority of the supernatant (about 900. mu.l) was discarded, the remaining 100. mu.l of the bacterial solution was applied uniformly to YEB solid medium containing kanamycin and rifamycin (concentrations of 50. mu.g/mL, respectively), and the mixture was cultured in an inverted state at 28 ℃ for 48 hours.
(2) Genetic transformation of cultivated rice embryogenic callus
Using young embryo of cultivated rice as genetic transformation material, inducing and culturing embryogenic callus, and culturing callus and agrobacteriumThe whole agrobacterium-mediated genetic transformation of the cultivated rice is completed through the processes of co-culture, screening and subculture of resistant callus, regeneration into a complete plant and the like. The obtained transgenic cultivated rice seedling is planted in the soil of a greenhouse (artificial climate chamber) and subjected to transgenic identification and selfing passage, and finally the seedling containing the transgenic cultivated rice can be obtainedSH4Transgenic oryza sativa lines with gene silencing elements.
In the invention, the specific operation flow of the step (4) is as follows:
(1) the transgenic cultivated rice line is artificially hybridized with weedy rice to obtain a hybrid F1Generation;
(2) the hybridization F1Inbreeding the generations to obtain hybrid F2Generation group;
(3) for the hybridization F1Generation population and hybrid F2The generation population is subjected to transgenic component identification, which can be classified as containing transgenes (F)1+，F2+) And no transgene (F)1-，F2-) Two separate populations of (a);
(4) subjecting the hybrid F1Generation population and hybrid F2Planting the generation group in the field, and performing seed falling property in the mature period of the seeds (SHI) Detection assay of, if presentSH4Transgenic populations of gene silencing elements (F)1+，F2+) In comparison with the transgenic-free population (F)1-，F2-) Is/are as followsSHIA low value indicatesSH4The gene silencing reduces the seed shattering of the filial generation of the cultivated rice and the weedy rice.
The specific method for measuring the seed shattering performance comprises the following steps: randomly selecting 3-5 plants in the mature period of the filial generation plants of the transgenic cultivated rice and the weedy rice, randomly selecting 10-20 fully mature and full seeds on each plant, and measuring the maximum tensile force required to be born by the seeds in the falling process one by using a digital display tensile meterBTSThe unit is gram force (gf). Calculating the average of the tensile force values required for all seed fallingBTS ave And calculating the seed shattering index by using a mathematical model formulaSHIThe unit is percentage (%), and the calculation method is as follows:
the invention has the advantages that:
the construction of artificial microRNA gene silencing element or RNA interference gene silencing element with hairpin structure and plant expression promoter expressed in rice seedling layer tissue to form chimeric gene, and the chimeric gene is introduced into rice through genetic transformation to form gene containingSH4Transgenic oryza sativa in which the element is silenced. The compounds obtained by this processSH4The risk of transgene spread is reduced by reduced seed shattering once the oryza sativa transgene of the gene silencing element is transferred to the weedy rice population by gene drift; at the same time, compriseSH4The seed shattering property and the diffusion capability of the weedy rice group of the gene silencing element are greatly reduced, so that the characters with reduced shattering property can be transferred to the weedy rice group through gene drift from the cultivated rice to the weedy rice, and the weedy rice diffusion control function is realized.
Therefore, the application of the invention can greatly reduce the environmental safety risk and economic loss possibly caused after the transgene escapes to the weedy rice, and simultaneously achieve the purpose of reducing the spread of the weedy rice. Has good application prospect and economic value in the fields of safely using transgenic cultivated rice by utilizing biotechnology, controlling weed rice harm and the like.
Drawings
FIG. 1 is a schematic view of an embodimentSH4Filial generation F of gene silencing cultivated rice and weedy rice1(A) And F2(B) The results of the seed shatter test of (1), white bars in the figure indicate the absence ofSH4Progeny of crosses containing gene silencing elements, black barsSH4Progeny of a cross of the gene silencing element. From this figure it can be seen thatSH4The gene silencing obviously reduces the seed falling property of filial generations of the cultivated rice and the weedy rice.
Detailed Description
By the method of the present inventionSH4The gene silencing technology reduces the seed falling performance of filial generation seeds of the cultivated rice and the weedy riceAnd (3) testing: the tests were carried out from 3 months to 11 months in 2012 to 11 months in 2015, in laboratories and test fields of the institute of biotechnology, academy of agricultural sciences, fujian province, and the cultivar of the tested oryza sativa was Minghui-86 (MH 86) and the weedy rice was Wd 1292.
The specific test design and method are as follows:
(one) SynthesisSH4Gene silencing element:
(1) the method for synthesizing the artificial microRNA gene silencing element comprises the following steps: rice shattering genes were cultivated on a Web MicroRNA Designer (WMD) on-line tool platform (http:// WMD3. weightelworld. org)SH4The genome sequence (LOC _ Os04g57530.1, TIGR oryza sativa whole genome annotation database) is taken as a target sequence, and one gene capable of silencing is screenedSH4The most suitable artificial microRNA fragment of the gene has the nucleotide sequence as follows: TGTATTTACAAGGTCGCACGG, (SEQ. ID. NO. 9), and designated amiRNA-sh1, the target sequence of this fragment being located in oryza sativaSH4The nucleotide sequence from-137 to-117 of the upstream untranslated region of the 5' end of the gene is CCGTGCGGCCTTGTAAATACG (SEQ. ID. NO. 10). Then, using the endogenous microRNA precursor osa-MIR528 of cultivated rice as a template (from pNW55 vector), designing an artificial microRNA specific primer pair (Table 3) by a WMD online tool, replacing the artificial microRNA fragment with a corresponding gene specific silent fragment in the osa-MIR528 precursor according to the overlapping PCR steps (Table 1), and synthesizing the specific silent fragmentSH4Artificial microRNA silencing elements of genes:
PCR reaction systems for overlapping PCR steps a-c:
PCR reaction procedure:
table 3 overlapping PCR primers for constructing artificial microRNA gene silencing element
After the reaction of steps a-c of the overlap PCR is completed, the resulting product is subjected to 2% (v/w) agarose gel electrophoresis and tapping recovery, and the corresponding target fragment is separated and purified. And dissolving the recovered a-c fragment in water to be used as a template for the final step d of the reaction for synthesizing the artificial microRNA fragment.
And d, a PCR reaction system of the reaction step d:
the PCR reaction procedure was as follows:
the PCR reaction product of reaction step d was isolated and purified on a 1% (v/w) agarose gel. Finally obtained product containsSH4The artificial microRNA gene silencing element of the gene silencing fragment is named as ShTM-1.
(2) The method for synthesizing the RNA interference gene silencing element with the hairpin structure comprises the following steps: planting riceSH4The full-length sequence of the gene cDNA was segmented into consecutive short nucleotide fragments of about 100-300 base pairs in length and these short fragments were aligned for online BLAST sequence homology on the NCBI website (http:// www.ncbi.nlm.nih.gov /) with the sequence database of the known Expressed Sequence Tag (EST) from cultivated rice. The test is carried outSH4The 5' untranslated region and the (-137 to 26) exon in the cDNA sequence of the gene defined a continuous nucleotide fragment (sh 4 ri-3) of 163 base pairs in length, and based on the sequence of sh4ri-3, a pair of primers was designed:
sh4ri3-ma：5’-GGTACCACTAGTCCGTGCGGCCTTGTAAA（SEQ.ID.NO.7）
sh4ri3-mb:5’-GGATCCGAGCTCAGCCGTTCTTCCAGCAGTAG（SEQ.ID.NO.8）
for synthetic specific silencingSH4Having hairpin structure of geneRNA interference gene silencing elements. SpeI and KpnI cleavage sites (underlined bases in sh4ri 3-ma) and SacI and BamHI cleavage sites (underlined bases in sh4ri 3-mb), respectively, were added sequentially on both sides of the sh4ri3-ma and sh4ri3-mb primer pairs to ligate the RNA interference fragment to a specific cloning site in the pTCK303 vector to form a hairpin structure. Then PCR is carried out to synthesize the RNA interference fragment, and the PCR reaction system and the reaction program are as follows:
and (3) PCR reaction system:
PCR reaction procedure:
the PCR reaction product was separated and purified on 1% (v/w) agarose gel to obtain a DNA fragment againstSH4RNA of the gene interferes with gene silencing fragments. Subsequently, the RNA interference gene silencing fragment is subjected to SpeI/SacI double enzyme digestion and is connected with a plant expression vector pTCK303 which is also subjected to SpeI/SacI double enzyme digestion to form an intermediate vector pTCK 303-a. Then, the same RNA interference gene silencing fragment is subjected to KpnI/BamHI double enzyme digestion and is connected with an intermediate vector pTCK303-a which is also subjected to KpnI/BamHI double enzyme digestion. Finally obtaining the specific silencingSH4RNA of the gene having a hairpin structure interferes with the gene silencing element and is named ShTM-2. The specific steps of the enzyme digestion and ligation reaction are as follows:
enzyme digestion reaction: preparing a vector plasmid to be subjected to (double) enzyme digestion or a PCR product into an enzyme digestion reaction system according to the following system:
the vector plasmid or the PCR product is subsequently cleaved at the optimum temperature for the restriction enzyme used (typically 37 ℃) for about 12 hours. The product obtained after completion of the cleavage reaction was separated on a 1% (v/w) agarose gel and the desired fragment was recovered for ligation.
Connecting reaction: using a ligation kit to obtain a vector plasmid large fragment and a small fragment or a PCR product obtained by enzyme digestion reaction;
mixing the components in a certain proportion, and carrying out ligation reaction for about 12 hours under the condition of water bath at 16 ℃ to obtain the recombinant plant expression vector plasmid.
Connecting a reaction system:
will be able to driveSH4Plant expression promoter with gene silencing element for high-efficiency expression in rice seedling abscission layer tissue, such as Ubi promoter or pSH4 promoter, and the likeSH4And (3) fusing the gene silencing elements to form a chimeric gene: in the test, a promoter Ubi from a maize ubiquitin gene is selected as a constitutive strong expression promoter.
And (III) introducing the chimeric gene into a cultivated rice explant through genetic transformation by using a plant expression vector such as pCXUN, pCXUN-osaMIR528, pCAMBIA1301, pTCK303 and the like to obtain a transgenic cultivated rice line:
SH4the gene silencing element ShTM-1 is directly connected with the oryza sativa transformation vector pCXUN cut by XcmI enzyme, and the cloned gene silencing element is driven by the Ubi promoterSH4The plant expression vector Ubi-ShTM-1 of the gene silencing fragment.SH4The gene silencing element ShTM-2 is integrated behind a Ubi promoter of a plant expression vector pTCK303 to obtain a gene silencing expression vector Ubi-ShTM-2;
the above-mentioned components are mixedSH4The gene silencing element and a plant expression promoter are fused to form a recombinant plant expression vector Ubi-ShTM-1 and Ubi-ShTM-2 of a chimeric gene, and the recombinant plant expression vector Ubi-ShTM-1 and the Ubi-ShTM-2 are introduced into a cultivated rice explant through genetic transformation to obtain transgenic cultivated rice strains TM-1 and TM-2, and the specific steps are as follows:
1. agrobacterium transformation, culture and identification
Mixing cultivated riceSH4Recombination of Gene silencing elementsThe plant expression vector plasmid is transferred into agrobacterium tumefaciens (A) by an electric shock transformation methodAgrobacteriumtumefaciens) Strain EHA 105. After the agrobacterium is shocked to absorb plasmids, 1000 mul of YEB culture medium (Agrobacterium rhizogenes culture medium) without antibiotics is added, shaking table is slowly shaken at 28 ℃ for 2 hours (100 rpm), the culture is centrifuged for a short time, the thalli is collected, most of supernatant (about 900 mul) is discarded, the rest 100 mul of bacterial liquid is uniformly coated on YEB solid culture medium containing kanamycin and rifampicin (the concentration is 50 mug/mL respectively), and the YEB solid culture medium is inverted and cultured at 28 ℃ for 48 hours;
2. genetic transformation of cultivated rice embryogenic callus
The whole agrobacterium-mediated genetic transformation of the cultivated rice is completed by taking the young embryo of the cultivated rice indica rice variety Minghui-86 as a genetic transformation material through the processes of embryogenic callus induction and culture, callus and agrobacterium co-culture, selection and subculture of resistant callus, regeneration into a complete plant and the like. The obtained transgenic cultivated rice seedlings are planted in soil of a greenhouse (artificial climate chamber) and subjected to transgenic identification and selfing passage. Finally obtain the product containingSH4Each transgenic oryza sativa line of the gene silencing element is TM-1 and TM-2, respectively.
(IV) carrying out artificial hybridization on the transgenic cultivated rice line and the weedy rice to obtain a hybrid F1Generation group, hybrid F1The generation group is bred by self-crossing to obtain a hybrid F2And (4) generation groups. By pairing hybrids F1Generation population and hybrid F2The generation group is subjected to transgenic component identification, and can be classified as containingSH4Transgenic populations of gene silencing elements (F)1+，F2+) And a population without transgenes (F)1-，F2-). Hybridization F1Generation population and hybrid F2The generation group is planted in the field, the seed falling property is measured and calculated in the plant mature period (SHI). When it containsSH4Of transgenic populations of gene silencing elementsSHIValue ratio of the population without transgeneSHIA low value indicatesSH4The gene silencing element reduces the seed falling property of filial generation of the cultivated rice and the weedy rice, thereby establishing and utilizingSH4Gene silencing elementA technique for reducing the seed shattering property of filial generation seeds of cultivated rice and weedy rice. The method comprises the following specific steps:
(1) the transgenic cultivated rice line is artificially hybridized with weedy rice to obtain a hybrid F1Generation: selecting a composition containing differentSH4Transgenic oryza sativa (TM-1 and TM-2) with gene silencing elements as male parent and weedy rice (Wd 1292) as female parent (Table 4) were artificially crossed to obtain 2 combined crosses F1Generation group; and setting hybrid F of cultivated rice MH86 (male parent) and weedy rice Wd12921Generations served as non-transgenic controls (table 4, table 5).
TABLE 4 parental test materials for oryza sativa and oryza sativa for artificial hybridization
(2) Hybridization F1Inbreeding the generations to obtain hybrid F2The generation group comprises the following specific methods: the obtained hybrid F1And (3) germinating about 100 seeds and raising seedlings on a seedbed, and taking leaves to identify transgenic components when the seedlings start to enter a tillering stage (about 30 days). Then, hybrid F without transgene1Generation (hybrid male parent MH 86) and identified hybrid F containing transgene1And (3) generation (the hybrid male parents are TM-1 and TM-2), independent planting is carried out according to cells, 5 plants are planted in each row, 5 rows are planted, 25 single plants are counted, the plant spacing is set to be 20 cm, each combination is set to be repeated for 5 times, all the cells are arranged in the field according to a completely random scheme, and the cell spacing is 50 cm. After about 2 months, hybrid F1Flowering and selfing, measuring and calculating F when the seeds are naturally mature1Seed shattering of generations (SHI) Value, the harvested seed is hybrid F2Generation population (table 5).
TABLE 5 transgenic oryza sativa and weedy rice artificial hybrid progeny F1And F2Experimental group
F1+And F2+Representing the presence of the transgene, F1-And F2-Indicates that no transgene was contained.
(3) Hybridization F2The generation group can be classified as containing transgenes (F) through seed germination, seedling raising and transgenic component identification2+) And no transgene (F)2-) Two separate populations of (i.e. F)2+ (TM-1)、F2+(TM-2) population and F2- (TM-1)、F2-(TM-2) population. All hybrids F2The generation groups are respectively used as independent experimental combinations and are planted separately according to the cells, 5 plants are planted in each row, 2 rows are planted, 10 single plants are counted, the plant spacing is set to be 20 cm, each combination is set to be repeated for 5 times, all the cells are arranged in the field according to a completely random scheme, and the cell spacing is 50 cm. After about 2 months, hybrid F2Flowering and selfing, measuring and counting hybrid F when the seeds are naturally mature2Seed shattering of generations (SHI) Value, then harvest F2Seeds of generations.
The specific method for measuring the seed shattering performance comprises the following steps: randomly selecting 3-5 plants in the mature period of the filial generation plants of the transgenic cultivated rice and the weedy rice, randomly selecting 10-20 fully mature and full seeds on each plant, and measuring the maximum tensile force required to be born by the seeds in the falling process one by using a digital display tensile meterBTSThe unit is gram force (gf). Calculating the average of the tensile force values required for all seed fallingBTS ave And calculating the seed shattering index by using a mathematical model formulaSHIThe unit is percentage (%), and the calculation method is as follows:
。
TABLE 6SH4Gene silencing hybrid F of cultivated rice and weedy rice Wd12921Generation and F2Average of the tensile forces required to shatter seeds of a population: (BTS ave ) And the index of seed shatterability: (SHI）
F1+And F2+Representing the presence of the transgene, F1-And F2-Indicates that no transgene was contained.
To the obtainedSH4Gene silencing cultivated rice line and weedy rice Wd1292 hybrid F1Generation and hybridization F2Seed shatter measurements of the passage population (table 6, figure 1) indicated that:SH4gene silencing hybrid F of cultivated rice and weedy rice Wd12921Generation and hybridization F2Transgenic-containing population of the progeny population (F)1+And F2+) In comparison with the transgenic-free population (F)1-And F2-) Seed shattering property of (A)SHI) The values show a significant reduction (table 6, fig. 1). Therefore, the temperature of the molten metal is controlled,SH4the gene silencing can obviously reduce the seed shattering property of the weedy rice, thereby reducing the risk of transgene diffusion; at the same time, compriseSH4The seed shattering property and the diffusion capability of the weedy rice group of the gene silencing element are greatly reduced, so that the character of reduced shattering property can be transferred to the weedy rice group through gene drift from the cultivated rice to the weedy rice, and the weedy rice can be controlled to diffuse.
A sequence table:
SEQ.ID.NO.1：AGTGTATTTACAAGGTCGCACGGCAGGAGATTCAGTTTGA
SEQ.ID.NO.2：TGCCGTGCGACCTTGTAAATACACTGCTGCTGCTACAGCC
SEQ.ID.NO.3：CTCCGTGGGACGTTGTAAATACATTCCTGCTGCTAGGCTG
SEQ.ID.NO.4：AATGTATTTACAACGTCCCACGGAGAGAGGCAAAAGTGAA
SEQ.ID.NO.5：CTGCAAGGCGATTAAGTTGGGTAAC
SEQ.ID.NO.6：GCGGATAACAATTTCACACAGGAAACAG
SEQ.ID.NO.7： GGTACCACTAGTCCGTGCGGCCTTGTAAA
SEQ.ID.NO.8：GGATCCGAGCTCAGCCGTTCTTCCAGCAGTAG
SEQ.ID.NO.9：TGTATTTACAAGGTCGCACGG
SEQ.ID.NO.10：CCGTGCGGCCTTGTAAATACG
SEQUENCE LISTING
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<120> method for reducing seed shattering of filial generation of oryza sativa and oryza sativa by SH4 gene silencing
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Claims (3)
1. Use ofSH4A method for reducing seed shattering of cultivated rice and hybrid progeny of cultivated rice and weedy rice by using a gene silencing element, characterized in that the shattering gene in the cultivated rice is made by using a gene silencing techniqueSH4Loss of function, silentSH4The gene gradually infiltrates into the weedy rice group through hybridization, can reduce the seed shattering property of the hybrid progeny group of the cultivated rice and the weedy rice, and comprises the following specific steps:
(1) synthesis ofSH4A gene silencing element;
(2) will be able to driveSH4Plant expression promoter with gene silencing element efficiently expressed in rice seedling abscission layer tissue, and the promoterSH4Gene silencing element fusionSynthesizing to form a chimeric gene;
(3) introducing the chimeric gene into cultivated rice by a transgenic method by using a plant expression vector to obtain a transgenic cultivated rice strain;
(4) simulating the process of the cultivated rice gene drifting to the weedy rice, and artificially hybridizing the transgenic cultivated rice line and the weedy rice to obtain a hybrid F1Generation group, hybrid F1The generation group is bred by self-crossing to obtain a hybrid F2Generation group; by pairing hybrids F1Generation population and hybrid F2Identifying transgenic components in the generation group, and classifying into containingSH4Transgenic population of gene silencing elements F1+，F2+And population F without transgenes1-，F2-(ii) a For hybridization F1Generation population and hybrid F2Planting in the field of the generation group, measuring and calculating seed falling property in plant maturation periodSHI(ii) a When it containsSH4Of transgenic populations of gene silencing elementsSHIValue ratio of the population without transgeneSHIA low value, i.e. indicatingSH4The gene silencing element reduces the seed falling property of filial generations of the cultivated rice and the weedy rice;
synthesis in step (1)SH4The silencing elements in gene silencing elements are of two types, respectively:
(1) the artificial microRNA gene silencing element is synthesized by the method that the artificial microRNA gene silencing element contains and encodes cultivated riceSH4A nucleotide sequence in which the difference of the continuous 21 nucleotide sequences in the cDNA nucleotide sequence of the gene is not more than 2 bases and a nucleotide sequence complementary thereto, and replaces the endogenous gene of oryza sativaosaMIR528The corresponding gene silencing nucleotide sequence of (a), capable of forming a double-stranded RNA molecule in a plant;
(2) RNA interference gene silencing element with hairpin structure is synthesized by using DNA encoding cultivated riceSH4The cDNA nucleotide sequence of the gene has 100 to 300 continuous nucleotide sequences and a nucleotide sequence complementary with the continuous nucleotide sequences, is separated by an intron in a plant expression vector pTCK303 and can form a double-stranded RNA molecule in a plant body;
the method for measuring seed shattering performance in step (4)The method comprises the following steps: randomly selecting 3-5 plants in the mature period of the filial generation plants of the transgenic cultivated rice and the weedy rice, randomly selecting 10-20 fully mature and full seeds on each plant, and measuring the maximum tensile force required to be born by the seeds in the falling process one by using a digital display tensile meterBTSIn units of gram force; calculating the average of the tensile force values required for all seed fallingBTS ave And calculating the seed shattering index by using the following mathematical model formulaSHIThe unit is as follows:
2. the method according to claim 1, wherein the plant expression promoter of step (2) comprises a promoter with strong constitutive expression or a promoter with specific expression in a abscission layer; wherein the constitutive strong expression promoter is selected from the promoter Ubi from the ubiquitin gene of maize, and the promoter for the specific expression of the abscission layer tissue is selected from the gene of the shatter of cultivated riceSH4The promoter of (3), pSH 4.
3. The method of claim 1, wherein the plant expression vector of step (3) is pCXUN, pCXUN-osaMIR528, pCAMBIA1301 or pTCK 303.
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