Method and application of genetically modified seed sorting by regulating seed weight

Abstract

The present invention discloses a method for sorting transgenic seeds by regulating seed weight and its application, belonging to the field of biotechnology. The present invention utilizes the difference in rice grain weight to improve the accuracy and efficiency of rice sterile line and maintainer line seed sorting, which has important theoretical and practical significance for the promotion and application of the third-generation hybrid rice technology.

Description

Technical Field

[0001] The present invention belongs to the field of plant biotechnology, and specifically relates to a new method for sorting transgenic and non-transgenic seeds, which regulates starch synthesis of rice seeds by constructing an expression vector, thereby controlling the morphology and weight of the seeds, and then sorting transgenic and non-transgenic seeds by weight difference by air sorting and gravity sorting. The present invention is applied to the third-generation hybrid rice technology to achieve efficient and accurate sorting of maintainer line and sterile line seeds. Background Art

[0002] Rice is an important food crop in my country. Since the 1980s, hybrid rice breeding technology based on male sterility has greatly improved the yield of rice per unit area, playing a key role in ensuring my country's food security. The third-generation hybrid rice technology is centered on molecular design. It is a new generation of hybrid rice technology that has been successfully applied after the "three-line method" based on nuclear-cytoplasmic interaction pollen sterility and the "two-line method" based on photothermosensitive nuclear sterility. This technology is also called "intelligent sterile hybrid breeding technology". This technology is based on genetically stable recessive male nuclear sterile materials, and the fertility restoration gene, pollen inactivation gene and sorting marker gene corresponding to the sterile material are introduced as tightly linked elements to form a maintenance line. The fertility restoration gene in the introduced element restores the fertility of the maintainer line. The presence of the introduced element causes the maintainer line to produce two types of male and female gametes in equal proportions during reproductive growth, one type does not contain the introduced element but only contains the male sterility mutation gene, and the other type contains both the introduced element and the male sterility mutation gene. The pollen containing the introduced element loses its activity under the action of the pollen inactivation gene, while the activity of the pollen without the introduced element is not affected. The introduced element does not affect female fertility, so self-pollination of the maintainer line can produce two types of seeds, namely, the sterile line without the introduced element and the maintainer line with the introduced element. These two types of seeds can be sorted by the sorting marker in the introduced element (such as red fluorescent protein). After sorting, the sterile line can be used for hybrid seed production, and the maintainer line is used for the reproduction of the sterile line.

[0003] Sorting markers are the main basis for distinguishing between maintainer and sterile seeds. Sorting between maintainer and sterile seeds is essentially sorting between transgenic and non-transgenic seeds. In existing production applications, the red fluorescent protein gene DsRed2 is usually used as a sorting marker gene. DsRed2 driven by the barley promoter Ltp2 with a 35s enhancer can be expressed in the aleurone layer of seeds, making the aleurone layer appear pink. Under the irradiation of a suitable light source, DsRed2 can be effectively excited and emit bright red fluorescence. Special color sorting equipment combines an imaging system with a mechanical sorting system to achieve large-scale and efficient sorting of maintainer and sterile seeds based on red fluorescence. However, in actual production, stunted seeds often appear, resulting in weak fluorescence of transgenic seeds, and color sorting equipment cannot accurately sort them; in addition, the high design and maintenance costs of color sorting equipment also increase the production costs of breeding companies. Finding a new efficient and low-cost sorting method to distinguish between maintainer and sterile seeds is of great significance to the application and promotion of the third-generation hybrid rice technology. Summary of the invention

[0004] The present invention aims to solve the technical problems of high cost, insufficient efficiency and precision in the sorting of maintainer and sterile seeds in the third generation hybrid rice technology. The present invention provides a method for improving the precision and efficiency of rice sterile and maintainer seed sorting by utilizing the difference in rice grain weight, specifically, by specifically regulating the transcription level of key genes for starch synthesis in the endosperm of maintainer seeds through gene silencing technology, thereby reducing the accumulation of starch in the endosperm and reducing the specific gravity of maintainer seeds; and the sterile seeds develop normally and have full grains. The maintainer seeds obtained by this method have a lighter specific gravity than sterile seeds, can be distinguished by wind selection and gravity selection machines, and are suitable for mechanized seed production.

[0005] The technical solution proposed by the present invention is:

[0006] A method for improving the accuracy and efficiency of rice sterile line and maintainer line seed screening in the third generation hybrid rice technology by utilizing the difference in specific gravity of rice seeds, characterized in that:

[0007] a) Using transgenic technology, a DNA fragment (deoxyribonucleic acid) that can affect the accumulation of seed components and other key elements in the third-generation hybrid rice technology are introduced into male sterile rice lines and planted;

[0008] b) Collecting all seeds together and sorting the mixed seeds according to their specific gravity after harvesting;

[0009] c) After sorting, the seeds with heavier specific gravity are the sterile line, and the seeds with lighter specific gravity are the maintainer line.

[0010] In the above method, the method that can affect the accumulation of seed components includes but is not limited to interfering with the expression of key genes involved in starch synthesis, wherein the key genes involved in starch synthesis include but are not limited to OsAGPL2, OsAGPS2, etc. Further, in a specific embodiment, a nucleotide sequence having the function of reducing the transcription level of genes such as OsAGPL2 (ADP-GlucosePyrophosphorylase Large subunit 2, LOC_Os01g44220) or OsAGPS2 (ADP-GlucosePyrophosphorylase Small subunit2, LOC_Os08g25734) is selected. The interaction between OsAGPL2 and OsAGPS2 during seed development is very important for the accumulation of starch in rice endosperm. Mutating or silencing the OsAGPL2 gene can inhibit its expression, resulting in defects in starch synthesis and composite particle formation in endosperm cells during seed filling, which is specifically manifested as an incomplete filling phenotype, and the specific gravity of the seeds is reduced. Similarly, mutating or silencing the OsAGPS2 gene can also affect the specific gravity of seeds through similar principles. Mutating or silencing the two genes specifically in developing seeds does not affect other traits of the plant.

[0011] The technologies used for the above-mentioned gene mutation or silencing in the present invention include but are not limited to artificial microRNA, antisense RNAi, viral RNAi, hairpin RNAi and other technologies that inhibit gene transcription level or protein translation level by transgenic means.

[0012] In the above method, further, the vector also includes other elements, including sequences formed by linkage of fertility restoration elements and nucleotide sequences with the function of inactivating transgenic pollen. The fertility restoration elements include but are not limited to recessive nuclear male sterility genes such as OsNP1; the nucleotide sequences with the function of inactivating transgenic pollen include but are not limited to a fusion sequence consisting of an amyloplast signal peptide coding sequence and an α-amylase gene, wherein the amyloplast signal peptide includes but is not limited to ASP1 derived from rice, BT1 derived from corn, etc., and the α-amylase gene includes but is not limited to genes such as OsAA derived from rice, ZM-AA1 derived from corn, and ZM-AA2 derived from corn.

[0013] In the above method, further, the sorting method includes but is not limited to gravity selection and air selection or a combination of gravity selection / air selection. Furthermore, a vertical airflow air separator can be used.

[0014] Specifically, the present invention provides a vector comprising a nucleotide sequence that can affect the accumulation of seed components in an embodiment, and the nucleotide sequence can be selected from the following:

[0015] a) a first expression cassette comprising a fertility restorer gene;

[0016] b) a second expression cassette containing a DNA fragment that inactivates the transgenic pollen;

[0017] c) A third expression cassette comprising a DNA segment that can affect the accumulation of seed components.

[0018] The fertility restorer gene includes but is not limited to OsNP1, etc. More specifically, the nucleotide sequence of OsNP1 is shown in SEQ ID NO: 5, comprising upstream 2044bp, 3040bp gene sequences and downstream 448bp.

[0019] The DNA fragment that can inactivate transgenic pollen includes, but is not limited to, a fusion sequence consisting of an amyloplast signal peptide coding sequence and an α-amylase gene, wherein the amyloplast signal peptide includes, but is not limited to, ASP1 from rice, BT1 from corn, etc., and the α-amylase gene includes, but is not limited to, OsAA gene from rice, ZM-AA1 gene from corn, ZM-AA2 gene from corn, etc. More specifically, the amyloplast signal peptide ASP1 coding sequence and the α-amylase gene OsAA in the fusion sequence are derived from rice, and the nucleotide sequence thereof is shown in SEQ ID No:7.

[0020] The second expression cassette has a pollen-specific expression promoter, and preferably the pollen-specific expression promoter is LSP3, whose nucleotide sequence is shown in SEQ ID NO:6.

[0021] The second expression cassette has a terminator, and preferably the terminator is In2-1, whose nucleotide sequence is shown in SEQ ID NO:8.

[0022] Among them, the ways in which the third expression box can affect the accumulation of seed components include but are not limited to interfering with the expression of key genes involved in starch synthesis, wherein the key genes involved in starch synthesis include but are not limited to OsAGPL2, OsAGPS2, etc.

[0023] The interference with the expression of key genes involved in starch synthesis includes but is not limited to mutation or silencing of key genes, and the technologies used include but are not limited to artificial microRNA, antisense RNAi, viral RNAi, hairpin RNAi and other technologies that inhibit gene transcription levels or protein translation levels through transgenic means.

[0024] The nucleotide sequence capable of affecting the accumulation of seed components is preferably the OsAGPL2 interference fragment miRAGPL2 or the OsAGPS2 interference fragment miRAGPS2, and the nucleotide sequences thereof are shown in SEQ ID NO: 2 or 3, respectively.

[0025] The third expression cassette has an endosperm-specific promoter, preferably the promoter is the rice-derived promoter P44220, whose nucleotide sequence is shown in SEQ ID No: 1. The third expression cassette has a terminator, preferably the terminator is PIN II TERM, whose nucleotide sequence is shown in SEQ ID NO: 4.

[0026] The beneficial effects of the present invention are:

[0027] The present invention specifically regulates the synthesis of starch in the endosperm of transgenic rice seeds, thereby affecting the seed specific gravity, and achieves accurate and efficient sorting of transgenic and non-transgenic seeds through air separation and gravity separation, and its beneficial effects include:

[0028] 1. Compared with the original third-generation hybrid rice technology in which maintainer and sterile line seeds are sorted according to red fluorescence, the sorting process in the present invention does not involve visual or image resolution, thus avoiding misjudgment caused by light obstruction or insufficient fluorescence intensity.

[0029] 2. The screening effect of the present invention on non-transgenic seeds can reach 100%. By using more stringent screening conditions, all transgenic seeds can be completely screened out, and at the same time, some underdeveloped or barren grains caused by diseases and insect pests in non-transgenic seeds can also be removed.

[0030] 3. Compared with the use of red fluorescent markers and matching color sorting equipment, the present invention enables the sorting of maintainer line and sterile line seeds in the third-generation hybrid rice technology to be completed using common agricultural machinery such as winnowing machines or grain cleaners with specific gravity selection functions, greatly reducing the requirements for equipment.

[0031] 4. The original third-generation hybrid rice technology used the maintainer line to pollinate the sterile line to produce sterile seeds in the actual seed production process, which had high seed production costs and low efficiency. However, the wind-selected Guangsan line can directly self-pollinate the maintainer line, combined with grain wind selection and gravity selection, to achieve mechanized seed production of maintainer line and sterile line seeds, greatly improving seed production efficiency and saving seed production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the structure of rice seed sorting marker expression vector p1300-miRAGPL2.

[0033] Figure 2The diagram shows the seed phenotype of transgenic rice positive plants, where the left diagram shows normal seeds produced by transgenic positive strains, and the right diagram shows seeds with reduced specific gravity produced by transgenic positive strains, bar=2mm.

[0034] Figure 3 Schematic diagram of the structure of the plant expression vector pOsFX.

[0035] Figure 4 The results of I2-KI staining of rice plant pollen, where the left picture shows the pollen staining result of wild-type plants, and the right picture shows the pollen staining result of transgenic positive plants, bar=250μm.

[0036] Figure 5 These are pictures of the wind selection effect of transgenic positive plant seeds, where the left picture shows the seeds before wind selection, the left side of the upper right picture shows normal seeds, the right side shows seeds with reduced specific gravity, and the lower right picture shows the seeds in the upper right picture with the shell removed, bar=2cm.

[0037] Figure 6 These are the growth phenotypes of plants produced from normal seeds produced from transgenic rice positive plants, where the left picture is a wild-type plant and the right picture is a plant produced from normal specific gravity seeds.

[0038] Figure 7 These are the growth phenotypes of plants produced from seeds with reduced specific gravity produced by transgenic rice positive plants, where the left picture is a wild-type plant and the right picture is a plant produced from seeds with reduced specific gravity.

[0039] Figure 8 Schematic diagram of the structure of the plant expression vector pOsFX2.

[0040] Fig. 9 The results of I2-KI staining of rice plant pollen, where the left picture shows the pollen staining result of wild-type plants, and the right picture shows the pollen staining result of transgenic positive plants, bar=250μm.

[0041] Fig.10 These are pictures of the wind selection effect of transgenic positive plant seeds, where the left picture shows the seeds before wind selection, the left side of the upper right picture shows normal seeds, the right side shows seeds with reduced specific gravity, and the lower right picture shows the seeds in the upper right picture with the shell removed, bar=2cm.

[0042] Fig.11 These are the growth phenotypes of plants produced from normal seeds produced from transgenic rice positive plants, where the left picture is a wild-type plant and the right picture is a plant produced from normal specific gravity seeds.

[0043] Fig.12 These are the growth phenotypes of plants produced from seeds with reduced specific gravity produced by transgenic rice positive plants, where the left picture is a wild-type plant and the right picture is a plant produced from seeds with reduced specific gravity. DETAILED DESCRIPTION

[0044] The following embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Any technician familiar with the art can use the above disclosed methods and technical contents to make many possible changes and modifications to the technical solutions of the present invention without departing from the spirit and technical solutions of the present invention, or modify them into equivalent embodiments of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still fall within the scope of protection of the technical solutions of the present invention.

[0045] Unless otherwise specified, the various technical means used in the examples are conventional means commonly used by those skilled in the art. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0046] Example 1: Construction of rice seed sorting marker expression vector p1300-miRAGPL2

[0047] By assembling the various elements of the miRAGPL2 expression cassette, the inventors constructed Figure 1 The p1300-miRAGPL2 vector shown in the figure. The expression cassette structure is P44220::miRAGPL2::PIN II TERM, which includes the promoter P44220 from the rice genome (shown in SEQ ID No: 1), the nucleotide sequence miRAGPL2 (shown in SEQ ID No: 2) having the function of interfering with the expression of the OsAGPL2 gene, and the terminator PIN II TERM (shown in SEQ ID No: 4) derived from potato. The above expression cassette was connected to the pCAMBIA1300 vector, and the plant expression vector p1300-miRAGPL2 was finally obtained after sequencing verification.

[0048] Example 2: Rice transformation and phenotypic investigation

[0049] Seeds of the wild-type rice Wuyunjing variety were used to induce callus tissue, and the plasmid p1300-miRAGPL2 obtained in Example 1 was transferred into the Agrobacterium Agl0 strain by electroporation. The callus tissue was genetically transformed by the Agrobacterium transformation method, and the seeds produced by the transgenic positive plants were observed. Figure 2As shown in the figure, the seeds of the transgenic positive strains showed phenotypic separation in terms of endosperm shape and specific gravity. Specifically, although there was no obvious difference in the appearance of all seeds, the endosperm of some seeds shrunk due to insufficient starch accumulation, resulting in a decrease in seed specific gravity, while the endosperm and specific gravity of other seeds were not affected. This shows that the rice expression vector p1300-miRAGPL2 can regulate the filling process of rice plants and ultimately affect the endosperm shape and specific gravity of transgenic seeds.

[0050] Example 3: Construction of rice expression vector pOsFX

[0051] Before constructing the rice plant expression vector, the inventors first transformed rice with each expression cassette in the expression vector separately, and further verified the function of each expression cassette. The results showed that each expression cassette could work well when transformed into rice separately, achieving the expected design effect.

[0052] Furthermore, the inventors constructed Figure 3 The rice expression vector pOsFX shown in the figure. As shown in the figure, the vector contains three expression cassettes, wherein the first expression cassette is the expression cassette of the rice male fertility gene OsNP1, which is the full-length sequence of the OsNP1 gene, including 2044bp and 3040bp upstream gene sequences and 448bp downstream, as shown in SEQ ID No:5 in the sequence table; the second expression cassette is the pollen inactivation function expression cassette LSP3::ASP1::OsAA::In2-1, which includes the pollen-specific expression promoter LSP3 from the rice genome (shown in SEQ ID No:6), the fusion sequence composed of the coding sequence of the amyloplast signal peptide ASP1 and the α-amylase gene OsAA (shown in SEQ ID No:7) and the terminator In2-1 from the corn genome (shown in SEQ ID No:8); the third expression cassette is the selection marker expression cassette P44220::miRAGPL2::PIN II TERM, which includes the promoter P44220 from the rice genome (shown in SEQ ID The invention relates to a novel protein-enhancing enzyme, comprising a miRNA, a nucleotide sequence having the function of interfering with the expression of the OsAGPL2 gene (shown in SEQ ID No: 1), a nucleotide sequence miRAGPL2 (shown in SEQ ID No: 2) having the function of interfering with the expression of the OsAGPL2 gene, and a terminator PIN II TERM (shown in SEQ ID No: 4) derived from potato.

[0053] The above three expression cassettes were connected into the pCAMBIA1300 vector, and the plant expression vector pOsFX was finally obtained after sequencing verification.

[0054] Example 4: Rice transformation

[0055] Seeds of rice plants with the genotype OsNP1 / osnp1 were used to induce callus tissue, and the plasmid pOsFX was transferred into the Agrobacterium Agl0 strain by electroporation. The callus tissue was genetically transformed by the Agrobacterium transformation method, and 25 transgenic positive plants were obtained, of which 11 had a background genotype of osnp1 recessive homozygous. The specific transformation recipient material was the rice variety Wuyunjing 7 background.

[0056] Example 5: Pollen fertility detection of transgenic rice plants

[0057] Analysis of the 25 transgenic positive rice plants obtained in Example 4 revealed that there was no obvious morphological difference between the transgenic positive plants and the non-transgenic control plants, but the pollen fertility was significantly different.

[0058] The transgenic plant materials obtained by transforming rice with the pOsFX construct and the wild-type rice control were tested for pollen dyeability. The method used was: during the flowering period of rice, single plants were randomly selected from the transgenic plants and the wild-type control plants, and a small flower from each plant was placed in the center of a slide, and a drop of 1% I2-KI solution was added. The anthers were crushed in the drop with tweezers to release the pollen, and the coverslip was covered. The staining was observed under a microscope. The dark blue pollen was fertile, and the pollen that could not be dyed was aborted.

[0059] The pollen stainability of transgenic plants was analyzed. Figure 4 As shown: the dyeable pollen of the control wild-type plants accounted for 98% to 100%; while in 5 randomly selected transgenic plants, the ratio of normal pollen to aborted pollen was close to 1:1, indicating that the rice expression vector pOsFX can achieve the expected pollen inactivation function.

[0060] Example 6: Separation and analysis of the specific gravity of seeds produced by transgenic rice plants

[0061] The T1 generation seeds of the transgenic rice plants described in Examples 4 and 5, whose background genotype was osnp1 recessive homozygous and whose ratio of normal pollen to sterile pollen was close to 1:1, were sorted according to their specific gravity. In this example, a vertical airflow air separator was used to sort the seeds. Since there was no significant difference in appearance and volume between the two types of seeds, the specific gravity of the maintainer seeds was reduced, and the specific gravity was significantly different from that of the sterile seeds (e.g. Figure 5 As shown in the figure, in a suitable wind field, the seeds of the retaining line rise with the airflow and are eventually discharged from the light material outlet, while the seeds of the sterile line fall under the action of gravity and are eventually discharged from the heavy material outlet. The results showed that the seeds of different specific gravities produced by these plants all showed a 1:1 separation ratio, indicating that the rice expression vector pOsFX can achieve the expected function of regulating the specific gravity of seeds.

[0062] Example 7: Fertility analysis of seeds of different specific gravities produced by transgenic rice plants

[0063] The two types of seeds sorted in Example 6 were planted and their fertility was examined. The results showed that all the seeds with normal specific gravity were sterile (such as Figure 6 As shown), all seeds with reduced specific gravity are fertile maintenance lines (as shown Figure 7 The results show that the expression of the various elements of the vector provided by the present invention as a whole can achieve the expected fertility restoration function, pollen inactivation function and seed screening marker function.

[0064] Example 8: Construction and effect analysis of rice expression vector pOsFX2

[0065] The miRAGPL2 (shown in SEQ ID No: 2) in Example 3 was replaced with miRAGPS2 (shown in SEQ ID No: 3) to construct the following Figure 8 The pOsFX2 vector verified by sequencing was used to transform rice according to the operation of Example 4, and the effect of the vector was analyzed according to the operation of Examples 5 to 7. Fig. 9 As shown in the figure, in the transgenic plants, the ratio of normal pollen to aborted pollen is close to 1:1, indicating that the rice expression vector pOsFX2 can achieve the expected pollen inactivation function. Fig.10 As shown in the figure, the seeds of different specific gravity produced by transgenic plants all showed a segregation ratio of 1:1, indicating that the rice expression vector pOsFX2 can achieve the expected function of regulating seed specific gravity. The seeds with normal specific gravity are all sterile lines (such as Fig.11 As shown), all seeds with reduced specific gravity are fertile maintenance lines (as shown Fig.12 ), indicating that the expression of each element of the vector provided by the present invention as a whole can achieve the expected fertility restoration function, pollen inactivation function and seed screening marker function.

[0066] The above examples show that the method provided by the present invention for improving the accuracy and efficiency of rice sterile and maintainer seed screening by utilizing the difference in specific gravity of rice seeds is feasible, with an actual utilization efficiency of 100%, no transgenic escape occurs, and biosafety can be ensured. Sequence Listing <110> South China Normal University Shenzhen Institute of Crop Molecular Design and Breeding Shenzhen Guangsanxi Agricultural Technology Co., Ltd. <120> Method and application of genetically modified seed sorting by regulating seed weight <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 2029 <212> DNA <213> Rice (Oryza sativa L.) <400> 1 aagctttatt gagctttgtg aatggacctt gcgtgagctc tgaaaccagc ttaccaccgt 60 tagttccagc tcggttttct ttttttcccc ttaaaggttt catgcgtatc gtgacagatg 120 ttacataatg acaaattccc cagctggagc acctttatcc ctgctgtttg catgaaatta 180 gcttgtcttg tagttccctc cagcaaaaag aagtctgaaa caaaacaaca tttcgaaaaa 240 aaggcatcca tgagttagca tttctacagt tgtctataga ggggaaggct gcacgacaaa 300 gtttccaggc ttggaaacaa cctcttatgt aaaatttttc gtatgtatca gatgatttgt 360 ttgcgttacg gcatctccac ctaacatcac cttcatcatg cgcctatggt ctttctcttg 420 cctgttttat acgtaaaatt ggaaacgaca gaaacttttg ccatctttat taaaggaagg 480 caaatatgca aatataggca tcaagatcac agttagtgga ttatcatctt tgtaggttaa 540 catgtcctac cccaggggag cttatactca agtactccat gcattttcat gaaatgagaa 600 aaaacgattt ttaagagaaa tgtactttct tgtatttatg ccaaatggca aggactgaaa 660 gggaaaaact aagaaaggga acgttacagt aaggctctgt ggggactggg gacttcagag 720 aaacgtgaac cctgcttcct tcctctgcat gaacataaca ccagaggttt ccagcctttc 780 acacagttgt tgatggcttc acacaattca tctctacctc ctgactcttt ataaggaccc 840 ccagcatcac cacaattgca caagtacagg cattagatcc acaagaacac ttgggcaggc 900 aagcacctct ttgatcttta agccgttgtt atgttctatt tctgagcata tggtttctag 960 ttatattctt tttcttcatt cgtttcatat ctttgaagtg ttgatgcaaa tgcagtgaac 1020 aactatcaac tgtgtactct ccaagtgaat gcgaataatc atttcctgtg agaattgtgg 1080 gctagataaa cgaacgaaat gctgttttat ctatgtcatg tgtggaaatt tagttaattt 1140 tccggtcttt ttatgcattg agatgggtat gctgtttttt tagttgggtc ccatcatctt 1200 gaaaattctt tcaaatttcc ttttctttat cctatataaa ggatagagaa ggcgtatgcc 1260 taggtgcacc aaccctgaaa gttttattct aattgcggga atggtttgta atttttgctt 1320 gttcaggttc tttttcgtgg cctttctttt ttttcccctt attttgctta gtctttcaca 1380 gtccaatttt tgggaagtag tatatcttag tttggtccta aggcaccatg ttgtactgca 1440 ggaaaaaa gagtaattgt attctgtttt ttccttgatt actatatccc tgttttaatt 1500 aattttgtgc ctttgttgct tgatgttgga acttcaatgc ccataattag tcatttgact 1560 tgttttgggt tttggtgcta tcttgagtgc cataggaaac tggtagaatt tagtaataat 1620 tttataga ctgaatgttg agcccaccac aaatggtttc cttctgtaca agtatttaat 1680 aactcaagca caggaaacat cagatctcta atctaaaggt taacaatggg ctcaagcagg 1740 agcagtagtt cagctctatc tgtatattta gaagggctgg atctacctgt ccaccagctt 1800 ttaatttac cctggcagct ggataacttc ttgtctgtta atttcattta gtgctgtgtt 1860 attttcttct tgttgttcag gatggatgct tttgaatttc tggaatttcg tattttgttc 1920 tatctcttta tgaaatgacg ttatggcaca ctttttctgc atattcttga tgaaataat 1980 tacctagtca tttttttagt tgcaggtttg tctgggactt tgagtaccc 2029 <210> 2 <211> 261 <212> DNA <213> Rice (Oryza sativa L.) <400> 2 tcggatccca gcagcagcca cagcaaaatt tggtttggga taggtaggtg ttatgttagg 60 tctggttttt tggctgtagc agcagcagtt atgctctaaa acggctctcc aggagattca 120 gtttgaagct ggacttcact tttgcctctc tgagaggcgt attagagcat aattcctgct 180 gctaggctgt tctgtggaag tttgcagagt ttatattatg ggtttaatcg tccatggcat 240 cagcatcagc agcggtaccg a 261 <210> 3 <211> 261 <212> DNA <213> Rice (Oryza sativa L.) <400> 3 tcggatccca gcagcagcca cagcaaaatt tggtttggga taggtaggtg ttatgttagg 60 tctggttttt tggctgtagc agcagcagta ttcttcttat aatgcccccc aggagattca 120 gtttgaagct ggacttcact tttgcctctc tggggggatt ttaagaagaa tattcctgct 180 gctaggctgt tctgtggaag tttgcagagt ttatattatg ggtttaatcg tccatggcat 240 cagcatcagc agcggtaccg a 261 <210> 4 <211> 349 <212> DNA <213> Potato (Solanum tuberosum L.) <400> 4 ttcgaacgcg taggtaccac atggttaacc tagacttgtc catcttctgg attggccaac 60 ttaattaatg tatgaaataa aaggatgcac acatagtgac atgctaatca ctataatgtg 120 ggcatcaaag ttgtgtgtta tgtgtaatta ctagttatct gaataaaaga gaaagagatc 180 atccatattt cttatcctaa atgaatgtca cgtgtcttta taattctttg atgaaccaga 240 tgcatttcat taaccaaatc catatacata taaatattaa tcatatataa ttaatatcaa 300 ttgggttagc aaaacaaatc tagtctaggt gtgttttgcg aatgcggcc 349 <210> 5 <211> 5533 <212> DNA <213> Rice (Oryza sativa L.) <400> 5 tcaattgaag aatttaccat ttgtcactct accaaaatac tagtgctcaa atgccactct 60 cccaaatatt cattcctaaa tgtcacccgg acctcaccac ttatgtttga atgaaaagtg 120 aggctgacat gtgcgttcgg atggcatttg ggaatgaaaa tttaggagag tggtatttga 180 gtgctatcat tttggtagtt gcaatatagta aattctcccc ctattttcta tgacactaaa 240 ctcagcccaa tactataaat tggactaaaa tatattttga aacggaggta gtagtataac 300 atatactatg tcctaaggct aaaaaggtgt gggggcgaag caaacattta cctagaaaac 360 tacgccataa tcaaaatgtg gcattacaaa gtgaagcaac taaccaaata accccaagta 420 atattagtaa gaaaatcccc caccagattt ttcaaattct caatcgcaag atctgtcatc 480 taaaatctac attttatctc caccctccaa tacaaacaaa tagtcaagcc ctagccacca 540 cttccacatc gctcctccta acctcaccct gtgctacgct gaaccaatca tgccctctgc 600 accaccacca tttagcctca atggcatctc cctcaacgct ggtgatgctc gccgacaagc 660 acctccatcg ccatccctat tgccatcatc ctccttgaga tggcttctca tgtgccatca 720 ccatttctta tgtccttacg ctgttgtctt cctcgtctct aatggtcttg tcgctgaatt 780 catgaccacc accacctaac caatctagcg aggtgcctga ccctattctc atcttccgtc 840 tcgagcaact caccatcacc taattacatg atcgggattc tctcgccata accactatcc 900 attgcctcg tagctacgac ggcgtcactg tcatctcgtt gccttcccaa tgccaccgtc 960 caccatcagg ccgctatctc cttagcctca ctctaagcgg aggaacctat gctctcta 1020 actggtcaga ccactgtcaa tgtctctgga cataatcaac attgttggag aggcagagag 1080 cagaaattgt gaggtagata taaaggttag atattgtaa gattttcctc ttaattttca 1140 atcgaatgtg gtttagggaa atggatattt ttttactag actggcgtat agcaatccga 1200 agacgaagaa agaaggagga ggagtaaaac acaaaaaaag aaaccaaccg tcttaaaact 1260 tacaacttta ccattgggca atacaactgc aatgggccaa gaagttaatt taaagttaag 1320 agcaaattca tttggaccac cttttgttac agatgcttca cttggacca cataccaccc 1380 atctctcttc tcgagcatga acaatctcga ttacattggc tcctactcat caataaactc 1440 tcacatatat atgtaaaacc attcatcggt atatgacaag ttatatatgg ataaaagagt 1500 tgaggatggg taagaataat aaccggtata aagtgagaac attgataaac atcgctaata 1560 aaagttcgtc tatagtaaaa tttactctaa attaaatca cctaatatttt tatatttt 1620 ttgtacaaat ggaccgtttc aatggggctt tatcagattt agttgagatg catacatggt 1680 aagcaccgtc ataatcttgc ccaagagctg acccaactca ttaaaattac gcttctttta 1740 cgacttaata aatcaagaag aaaccattga aatccagcct gccccgactg tctcgtaaca 1800 gaaaaataac tcagcaacga ctaaattatg attttaaaat ggcaaaaata tcaaagcacg 1860 ttcgaaacaa tcgcaagatt ggcaagtaaa ctctcctgct tgcttgctca caaccacatc 1920 agatcattga tcaatgtttc atcagctcat cacttctgca tgcatgttat attcttctca 1980 gggctccaca atttacaaag ctgctcgaag atcttctttg cagtgcaaag caatctgcaa 2040 gattattcaa gacatctact cttgatctac cattgagcta actccggata tataaacaga 2100 ccgaacgttt cgtcccaggg gaatgtgaaa gttagcgaat ttgcccggcg aaaatggcag 2160 cacttggccg cgcgagctcg tcggcgccgg tgcttgccgc cgccgccgcc gtgctcctct 2220 cgctctgcct cgccgcgctc tcggaagagc aaggtgcgta aacgttgcgt tgtatctttg 2280 cgttgatgcg tgttgcgtcg tcgtcgtgtt catggcgtgc gatggcgttg tgcagagcaa 2340 ctggagaacc tgcggttcgt gcggcacgcg caggacgcgc cgctggtgtc gagctacaac 2400 tacatcgtca tcggcggcgg cacggcgggg tgcccgctgg cggcgacgct gtcggagcac 2460 tcgcgcgtgc tgctgctgga gcgcggcggc ctgccgtacg ccaacatgtc gagcgagcag 2520 cacttcacgg acgcgctggc cgacacgtcg ccggcgtcgc cggcgcagcg gttcatctcg 2580 gaggacggcg tggtgaacgc ccgggcgcgg gtgctcggcg gcgggagctg cctcaacgcc 2640 gggttctaca cgcgggcgag caacgagtac gtgcgcgcct ccgggtggga cgcgcggctg 2700 gtgaactcgt cgtaccggtg ggtggagcgc tcgctggtgt tccgccccga cgtgccgccg 2760 tggcaggcgg cgctccgcga cgcgctgctc gaggtcggcg tcacgcccga caacggcttc 2820 accttcgacc acgtcaccgg caccaagatc ggcggcacca tcttcgacaa ctccggccag 2880 cgccacaccg ccgccgactt cctccgccac gcccgccccc gcggcctcac cgtcctcctc 2940 tacgccaccg tctcccgtat cctcttcaaa agccaaggta cacagctacg atgaaaatgg 3000 aaaatgtgct gtgcgccgaa gaagcttgac ctcacgacgg cgagcttttg ccatggcgtg 3060 cagacggcgt cccatacccg gtggcgtacg gggtggtgtt ctcggacccg ctgggggtgc 3120 agcaccgggt gtacctccgc gacggcgaca agaacgaggt gatcgtgtcg gcggggacgc 3180 tggggagccc gcagctgctg atgctgagcg gcgtcgggcc gcaggcgcac ctggaggcgc 3240 acggcatcga ggtgatcgtg gaccaaccca tggtcgggca gggcgtcgcc gacaacccga 3300 tgaactcggt gttcatcccg tcgccggtgc cggtggagct ctccctggtg caggtcgtcg 3360 gcatcacccg ctccggcagc ttcatcgagg gggtgagcgg gtcggagttc ggcatgccgg 3420 tgtcggacgg cgcgctccgg tgggcgcgca gcttcgggat gctgtcgccg cagacggggc 3480 agctcggcac gctgccgccg aagcagagga cgccggaggc gctgcagcgg gcggcggagg 3540 cgatgatgcg gctggacagg agggcgttcc ggggaggctt catcctggag aagatcctcg 3600 ggccggtgtc ctccggccac gtcgagctgc gaaccaccga cccgagggcg aacccgtcgg 3660 tgacgttcaa ctacttccgc gaggcagagg atctggagcg gtgcgtccat ggcatcgaga 3720 cgatcgagcg ggtgatccag tcgcgggcct tctccaactt cacctacgcc aacgcctccg 3780 tcgagtccat cttcaccgat tccgccact tccccgtca cctgctgccg cgccatgtca 3840 acgactcgcg ctcgccggag cagtactgca tggacaccgt catgaccacc tggcactacc 3900 acggcggctg ccatgtcggc gccgtcgtcg acgacgatta ccggtgttc ggggtgcagg 3960 ggctcagggt gatcgacagc tccaccttca agtactcccc cggcaccac cctcaggcca 4020 ccgtcatgat gctcggcagg taactggcat cattttagct catgaagtg cattgccatg 4080 agtaacaaca cactaacagt atagttttca atatggacac tgggcaggta tatgggtgtg 4140 aagattcagt ccgagagatg gagaatga tgacaaag atatttcgt ttcaggagca 4200 aaaaaatgca tgtattca ggaaaagaaa atgttcaact gtctttagag tttagagtag 4260 attttatttg cacccactta atttttactc ttcttagac ataggttcag tatctgcttg 4320 ttgattgt aaccttgaag aagcattgca aaaacaaagc ggaacttat gttaccaagg 4380 gcatgacgaa gaataatg gattagattt cattgacact tagaaatgg aaccagcaa 4440 tcaggctga aaatattac actagaaact tattttaatg gctttacatg tcgctacata 4500 cttaaatcaa tcaaagttgc taccaaagcc atgttcccta aacagagggt tccgggcttt 4560 caaacattct taatcttcta tacattgata aaaagtatac ataaaaagaa aacctattaa 4620 gatggaaatg ttgaattctc ttaagaaagg cataaaaaat gcagggtaat aaccttttct 4680 tgtcatgtcc tacttggttt caacctatat tgctagcaaa attttcacgc tatctataac 4740 ctcaggaagc agaatagctc agaatcacta gcaattgtat acttacagat aatcgaaatt 4800 catattgtga ggttttcttg gatggctcgg aatgatccac agatgttacc acccatgtct 4860 gggaatgacg tacatcctgg caagggtttc accttgccct tacggtcaac acttacaggg 4920 tacttcatta ggtggcctct catctcagtc acctcattat caacaaatcg ttcccagttc 4980 tgttcaccga tttggcgaac tcgtctcatg cattccatgg tctctggata gttgaagcct 5040 tcctcaacca ctccaatatg ctccgcccaa agagacattc tgtacccata aatctacaca 5100 tgaaatcagc agatttgaat tagagtgtgg ttctagaaat atcagtcatc ctaaaatagg 5160 tatatccctc aaaccaagag acaactagtc gagattcctt cacagttgta cagggctatt 5220 tgaaatatga ctgtacttga tatgcaactt tttctctatt tctacaaata catttgtatt 5280 ttccatacat ttgtagctac aatctggtcc caccatgatg gatggtgtag atagctctct 5340 ttttcttcaa ttaacatatt cttgagaatg aatggtagct tctctttttt tcttccaatt 5400 aacatattct taagagtgag tggtagcttt ttttagcctt atctaaaaaa atatgattga 5460 tagggtacga atttcccttt aggttactct gccaaaacaa aagataattg caacaaatat 5520 atatggctgg att 5533 <210> 6 <211> 2501 <212> DNA <213> Rice (Oryza sativa L.) <400> 6 gggtgacggc aggtactgat atatagagtt tagggtcgtg caaccaacca ttgacgcaac 60 cctaatgggt tccaacacga tacacggccc aaaggcccaa atacggtgac gcaacgccag 120 gacagattct agacgtcaac ttgcgagaag gctagttagc gatcagtcgc gcgcgcccct 180 ccccgcgcga ctggacacgt gtcgcgcgcg gagaggggac ggcggacgcg ccgcgctaag 240 aggagagaga gagagagagc aagttttccc tttttttgtt ttttcccttt ttttggtttc 300 tgttttgttg gttttcctt ttttttctttgg tttctctttt taaggtg gatatacaaag 360 tttgtattcg tatgtatacc aagtttgtat ttgtacgtat ataaactttg tatacgcgta 420 tgcaaatttt gtatacgttg tgtattttct agtttttt ttttatac gtatgtatat 480 aaagtttata ttcatcgtat aaagtttg tacgtcctat aaactttg tatatgtata 540 tattttcat gcattaaaaa tattattcata tatatatata tattatata tgaataccat 600 ggtatctaat gcataaaatc actcaaattt tttaaaattt tcaattgtg agtatatacc 660 tagttatat aattcgattc atacctatac ctatcaacaa aaaactca atttaacttt 720 atttcacaat ccatgattac atacctact aattatatgt atggatgcta tatacctaga 780 atcagatct aaaaaaca agttcaagtt cagttcaaac ttgctttgaa ctagttagta 840 aagtagttaa tgttaatacc taagtattg aaaagttttc attackcattt gattgggta 900 tatactcaat ttcaacaatg gtgcccgggc accatggagc accaaaaa ttatatat 960 fatherfatherfatherfatherfatherfatherfatherfatherfatherggt aaattaactc 1020 gtgctacatg gagtattggc tccaagattc aaattgagta tataaccaat ttgaatgagt 1080 atgaattttt tttaaatgtt ttaggtatga acattaactt ctttactaac aagtttgaac 1140 tgagtttgaa cttttttttt tgttagattt tgattctagg tatataacat ccaaacatat 1200 aattcgttag gtatgtaata atgaattgtg aaataaaagtt gagtttgagt tattttgttg 1260 ttaggtattg gtatgaatca aatttataga cctaggtata tactcacaat ttaaattttt 1320 taaaaaattt gagtgatttt gtgtgtttta taccttagag cccgggcacc atagagtccc 1380 atatgggtat ctatatatat atatacatat atatatatat acacattgga tatgttatct 1440 tcttagcttt ccatccatac aaagaaaacc ccaattcagt attaccttca atgtttaagt 1500 agttttgtga ttttaccaaa tagaattgct aaaagttttt tttgtttgga ttctatctat 1560 ttctgtgtca tcagatgcac accgagcgta aataaaatca cgcatgggg cgggtgatta 1620 aactcatgac catgcctacc aagggagcaa aattacccag atttcataat aattacatgc 1680 aagttgcata taagttataa aatttagttt aaaatataat tacactatat ttgcactata 1740 acgttatatt tatatattt ccacccaaaa agttatcgca gcacttttag agtggtcttt 1800 actataagtc atcgggatcg agtgacacca atgttaattg ttaattgtta gcctgtcaat 1860 atagatccac ccctaaacat aagaccaaac ttgccaagaa tatatctcgt tttaacacac 1920 tcttaatagc cgcgacttat ggtatactga caaacctcac aaaattctta ctgaaagcaa 1980 atcctccatg tgcgtgtgta cactaggatt taaactagat actgtgtacc caacgccacg 2040 gctaccccaa cgatgttatc ccctcactat actaatgtta ttctgggacc acctaacttt 2100 ctgcaaatat gaataataga catccaaatt taactcaaaa ttaaatcttc aatatttatt 2160 ttgcaactaa acccttgttc attgaacgca gcaacagaga aatctacgga tcttaccatt 2220 ctatttctct gtgaactaat tgcccagtac atgtctataa ataacatcac tcacctgcat 2280 gcgtccactc atccagcaac tgttctctg tgtaccgttt ctctcgccga ttgtgtaccg 2340 attcaattca aaattcgaa aacaacgcac catttttcgt aaatcataat tagtgctagc 2400 taagtcaatc ctgcaagcct atatattc gcttctctgc ataattagct gaatcgtctt 2460 cgttagcagt gagagtgaca gacgacacat agaaggtttt c 2501 <210> 7 <211> 1752 <212> DNA <213> Rice (Oryza sativa L.) <400> 7 atgcagccac tggtgagcac atccgccgca gcagctgcga ttccctcccc tcctcctccc 60 gcgctcactc ttccgcctcc acgccggcga atccccccgc ccctcaccgc cggagccctc 120 ctctgcaacc gcaccggctc cggccacgcc agcccgcggg gaagacgacc cctacccccg 180 ccggcgatgt cccacccgct ctcctccgaa cctcatgctt tggaacaaag ggcagtggta 240 atgggccaga tggtttcgga tgaaaatttt gaagagcagg ccgctcgtaa tggtggaatt 300 atcaagcacg gaagggaaat cctgttccag gcttttaact gggaatccca taagcacaac 360 tggtggagca atttggagga aaaagttgtt gacctagcac aatcggggtt tacatcagcg 420 tggctgcctc caccaacaca gtcattatct ccagaaggct atctgccgca gaacctgtac 480 tgcctcgact cttgttatgg ttccctccat gacctacaag cattgcttcg gaaaatgaaa 540 gaacacaatg taagagctat ggcagatgtt gttattaacc atcgagttgg gactactcaa 600 ggatcgaatg ggatgtataa tcgttatgat ggtatcccag tatcatggga tgaacatgct 660 gttacatctt gttctggtgg aaagggcaac gaaagtaccg gtgataactt cgatggggtt 720 cccaacatag accatacaca gccatttgta cggaaggata ttattgactg gttgatctgg 780 cttcgagaaa gcattggttt tcaagatttc cgctttgatt tcacgaaagg ttatgcagcg 840 aagtttgtga aagaatacat tgagcaatca aagcctcttt ttgcggtggg ggaatactgg 900 gatagctgtg aatatagccc ccctgattac cgtttgaact acaatcagga taaacacagg 960 cagagaatta tcaattggat ggatagcact ggaggacttt gtgctgcatt tgatttcaca 1020 acaaagggta ttcttcagga agctgtcaaa ggtgagttgt ggcgtttgcg tgaccctgaa 1080 ggaaagccgc ctggtgtcat gggctggtgg ccttcgagat cagtcacatt tgttgaaaat 1140 catgacacag gatccactca gggtcattgg ccattcccat ctgatcatat catggaggga 1200 tatgcttata tacttactca tcctggcatc cccacggtgt tctacgatca tttctatggc 1260 aaagatgatt ccttccatgg tggaattgca aaactgatgg agatcaggaa atgccaggac 1320 atacatagcc gctccgcagt caaaatcttg gaggccagct cagatctgta ctctgcaatc 1380 gttgacgata agttgtgcat gaagatcggg gacggctcct ggtgcccgag cggcccagag 1440 tggaagctgg cggcctctgg tgacagatat gctgtttggc acaagtagat ataccaacat 1500 atagcatttc tttctgctga acatgagata tgctatttct tcagagcccg aactgtatat 1560 gctagtacca tggtaattta atcatcttca atgcaatacc agagagcaaa ccacttgcaa 1620 tttatcacat gctcacagca gatagtgtaa actgtaatgc gaagtgtaaa ctgtaatgtg 1680 aatacgtata ctgtaatgct tgtgtgcata taggaataag agagtgttta tttatagaaa 1740 gcattcttgg ag 1752 <210> 8 <211> 325 <212> DNA <213> Zea mays L. <400> 8 gttgaatcgg tggaagacca ctcgtcagtg ttgagttgaa tgtttgatca ataaaatacg 60 gcaatgctgt aagggttgtt ttttatgcca ttgataatac actgtactgt tcagttgttg 120 aactctattt cttagccatg ccaagtgctt ttcttatttt gaataacatt acagcaaaaa 180 gttgaaagac aaaaaaaaaa acccccgaac agagtgcttt gggtcccaag ctactttaga 240 ctgtgttcgg cgttccccct aaatttctcc ccctatatct cactcacttg tcacatcagc 300 gttctctttc ccctatatct ccacg 325

Claims

1. A vector comprising a nucleotide sequence that can affect the accumulation of seed components, comprising the following nucleotide sequence: The first expression cassette contains the fertility restorer gene OsNP1, and the nucleotide sequence thereof is shown in SEQ ID NO: 5; The second expression cassette contains a fusion sequence consisting of ASP1 and OsAA that can inactivate transgenic pollen, and the nucleotide sequence thereof is shown in SEQ ID NO: 7; The third expression cassette contains the nucleotide sequence miRAGPL2 that can interfere with the expression function of the starch synthesis gene OsAGPL2 or the nucleotide sequence miRAGPS2 that can interfere with the expression function of the starch synthesis gene OsAGPS2; wherein the nucleotide sequence of miRAGPL2 or miRAGPS2 is shown in SEQ ID NO: 2 or 3, respectively.

2. The vector according to claim 1, wherein the second expression cassette is driven by a pollen-specific expression promoter, and the pollen-specific expression promoter is LSP3, whose nucleotide sequence is shown in SEQ ID NO:

6.

3. The vector according to claim 1, wherein the second expression cassette has a terminator, and the terminator is In2-1, whose nucleotide sequence is shown in SEQ ID NO:

8.

4. The vector according to claim 1, wherein the miRAGPL2 or miRAGPS2 is an artificial microRNA.

5. The vector according to claim 1, wherein the third expression cassette is driven by a promoter, the promoter is rice-derived promoter P44220, and its nucleotide sequence is shown in SEQ ID NO:

1. 6 . The vector according to claim 1 , wherein the third expression cassette has a terminator, and the terminator is PINII TERM, and its nucleotide sequence is shown in SEQ ID NO:

4.

7. A method for improving the accuracy and efficiency of rice sterile line and maintainer line seed screening in the third generation hybrid rice technology by utilizing the difference in specific gravity of rice seeds, characterized in that: (a) using transgenic technology to introduce the vector of claim 1 into rice and plant it; (b) harvesting all seeds together and sorting the harvested seeds according to their weight after harvesting; (c) After sorting, seeds without the vector are sterile and have a heavier specific gravity; seeds containing the vector are maintainer and have a lighter specific gravity.

8. The method according to claim 7, wherein the sorting is performed based on the seed specific gravity, including gravity sorting or wind sorting or a combination of gravity sorting and wind sorting.

Images