Sperm cell protection gene S19g-a7 and its application in improving hybrid fertility of rice and breeding of hybrid rice
By introducing the male gamete protection gene S19g-A7 into Asian rice, the problem of hybrid sterility caused by reproductive isolation between African and Asian rice was solved, realizing the restoration of fertility in interspecific hybrids of rice and the breeding of high-quality rice.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- SOUTH CHINA AGRICULTURAL UNIVERSITY
- Filing Date
- 2024-12-30
- Publication Date
- 2026-06-30
AI Technical Summary
African rice and Asian rice are reproductively isolated, resulting in highly sterile hybrids that make it difficult to utilize their hybrid vigor to increase rice yield.
The male gamete protection gene S19g-A7 was cloned and introduced into Asian rice for functional complementation. Overexpression was achieved through expression promoters or vectors, breaking the hybrid sterility mediated by the S19 locus and creating a rice hybrid compatible line.
It has achieved the restoration of pollen fertility between Asian and African rice, broken the interspecific hybrid sterility, created fertile hybrid rice, and carried out high-quality rice germplasm breeding by utilizing the heterosis of distant hybrids.
Smart Images
Figure CN122302018A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of genetic engineering technology. More specifically, it relates to a male gamete protection gene S19g-A7 at the S19 locus of interspecific hybrid sterility in rice and its application in improving hybrid fertility in rice and hybrid rice breeding. Background Technology
[0002] Utilizing the heterosis of different rice varieties to increase rice yield is a key strategy for improving grain production. Cultivated rice is divided into African cultivated rice (Oryza glaberrima, or simply African rice) and Asian cultivated rice (Oryza sativa, or simply Asian rice), which belong to different species.
[0003] African rice possesses a variety of excellent agronomic traits. It exhibits strong tolerance to environmental stresses such as heat, drought, and salinity, and demonstrates good resistance to various rice diseases. However, African rice shows significant reproductive isolation from other cultivated rice varieties, such as Asian rice, resulting in highly sterile hybrids. Therefore, finding ways to overcome the incompatibility problem between African rice and other rice varieties and creating compatible hybrid lines is of great importance. Summary of the Invention
[0004] This invention aims to improve the fertility of interspecific hybrids in rice. A new male gamete protection gene, S19g-A7, was cloned, and a method for creating rice hybrid compatible lines was developed using this gene and its encoded protein.
[0005] The first objective of this invention is to provide a rice male gamete protective protein S19g-A7.
[0006] The second objective of this invention is to provide a rice male gamete protection gene S19g-A7.
[0007] A third objective of this invention is to provide a reagent for overcoming interspecific hybrid sterility in rice.
[0008] A fourth object of the present invention is to provide the application of the above-mentioned protein S19g-A7, the above-mentioned gene S19g-A7, or the above-mentioned reagent.
[0009] The fifth objective of this invention is to provide a method for creating a rice hybrid compatible line.
[0010] The sixth objective of this invention is to provide a method for overcoming interspecific hybrid sterility and constructing fertile hybrid rice.
[0011] The above-mentioned objective of this invention is achieved through the following technical solution:
[0012] This invention provides a rice male gamete protective protein S19g-A7, the amino acid sequence of which is shown in SEQ ID NO.2.
[0013] This invention provides a rice male gamete protection gene S19g-A7, the nucleotide sequence of which is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2.
[0014] As an alternative implementation, the nucleotide sequence of the S19g-A7 gene is shown in SEQ ID NO.1: promoter region (1-2151): 2151bp, exon 1 region (2152-2297): 146bp, exon 2 region (2535-2899): 365bp, exon 3 region (4368-4603): 236bp, exon 4 region (9548-10105): 558bp, and terminator region (10106-13516): 3411bp.
[0015] The present invention provides a reagent for overcoming interspecific hybrid sterility in rice, characterized in that it includes an expression promoter of the gene S19g-A7 as described in claim 2.
[0016] As an alternative implementation, the expression promoter of the S19g-A7 gene includes an overexpression vector of the S19g-A7 gene.
[0017] This invention transforms African rice S19g-A7 into Asian rice for functional complementation. The resulting transgenic S19g-A7 rice... t The functionally complementary transformant exhibits fully fertile pollen fertility. When this complementary transformant is crossed with a near-isogenic line containing the S19g allele of African rice, the pollen fertility of the resulting F1 hybrid is partially restored, indicating that S19g-A7 is the pollen-protecting gene at the S19 locus. This complementary transformant is a compatible line for the Asian rice type S19 locus created in this invention and can be used to break hybrid sterility mediated by the heterozygous S19 locus.
[0018] Therefore, the present invention also provides the following application solutions:
[0019] Application of the above-mentioned protein S19g-A7, the above-mentioned gene S19g-A7, or the above-mentioned reagent in overcoming interspecific hybrid sterility in rice or improving the fertility of interspecific hybrids in rice.
[0020] Application of the above-mentioned protein S19g-A7, the above-mentioned gene S19g-A7, or the above-mentioned reagents in the creation of distant hybrid rice affinity materials.
[0021] Application of the above-mentioned protein S19g-A7, the above-mentioned gene S19g-A7, or the above-mentioned reagents in interspecific hybridization breeding of rice.
[0022] This invention provides a method for creating a rice hybrid compatible line by introducing the above-mentioned S19g-A7 gene into rice for functional complementation or overexpression.
[0023] As an alternative implementation, the functional complementarity or overexpression is achieved by using the S19g-A7 promoter, constitutive expression promoter, or pollen expression promoter to drive the expression of the S19g-A7 coding sequence.
[0024] As an alternative implementation, the constitutive promoter can be the CaMV35S promoter, and the pollen high expression promoter can be the OsPoll4 promoter or the OsAnth3 promoter.
[0025] As an alternative implementation, the functional complementarity or overexpression is achieved by using the S19g-A7 protein itself to express the S19g-A7 protein, including but not limited to the nucleotide sequence shown in SEQ ID NO.1.
[0026] As an alternative implementation scheme, the method for achieving S19g-A7 functional complementation is as follows: the S19g-A7 genome sequence is amplified using primers with nucleotide sequences as shown in SEQ ID NO.3-4, ligated into a binary expression vector, the constructed vector is transformed into Agrobacterium, and then infected with Asian rice.
[0027] As an alternative implementation scheme, the S19g-A7 functionally complementary plants obtained by the above method can be screened using primers with nucleotide sequences as shown in SEQ ID NO.5-6 to identify S19g-A7 transgenic positive plants, which are the Asian rice hybrid affinity lines.
[0028] The Asian rice S19 compatible line was crossed with African rice, and the resulting hybrid F1 (genotype gs / 7) t –) showed significantly improved pollen fertility, and offspring carried S19g-A7. t The pollen fertility of the transgenic homozygous S19 locus heterozygous plants was restored to full fertility, breaking the S19 locus-mediated hybrid male sterility phenotype.
[0029] This invention also provides a method for overcoming interspecific hybrid sterility and constructing fertile hybrid rice, wherein the rice hybrid compatible line created by the above method is hybridized with African rice to produce a fertile interspecific hybrid of Asian and African rice.
[0030] As an alternative implementation, the rice is Asian rice.
[0031] The present invention has the following beneficial effects:
[0032] This invention identified the key gene S19g-A7 at the S19 locus of hybrid sterility. The S19g-A7 gene was introduced into Asian rice (S19g-A7...). t Its pollen fertility is fully fertile, and S19g-A7 t The transgenic material was crossed with African rice to obtain hybrid F1 (genotype gs / 7). t The pollen fertility of the gene was significantly improved, and genetic analysis proved for the first time that S19g-A7 is the hybrid male gamete protection gene at the S19 locus. Therefore, transforming the S19g-A7 gene into Asian rice can create S19 locus compatible lines, which can break the interspecific hybrid sterility mediated by the S19 locus.
[0033] This invention develops a method for rapidly creating hybrid compatible lines that overcome hybrid sterility in Asian and African rice varieties. Based on gene functional complementation, this method rapidly creates hybrid compatible lines that overcome hybrid sterility in Asian and African rice varieties, providing genetic resources and breeding methods for utilizing distant hybrid vigor in high-quality rice germplasm breeding, and has significant application value. Attached Figure Description
[0034] Figure 1 The genome structure of the S19 locus is shown in Figure A (the genome structure of Asian rice type S19s and African rice type S19g on chromosome 3; Figure B is the gene structure of S19g-A7).
[0035] Figure 2 S19g-A7 t The results of the T0 generation transgenic detection are shown. M is the marker indicating the size of the band, CK+ is the positive control, and CK- is the negative control.
[0036] Figure 3 S19g-A7 t Results of pollen and spikelet fertility tests on the transformants (Figure A shows the pollen and spikelet fertility of control 9522; Figure B shows the fertility of S19g-A7). t Fertility of plant pollen and spikelets; pollen scale bar is 50 μm; spikelet scale bar is 5 cm; FF indicates fully fertile.
[0037] Figure 4 F1-S19g-A7 t The results of pollen and spikelet fertility tests (Figure A shows the pollen and spikelet fertility of the control F1; Figure B shows the fertility of F1-S19g-A7). t The fertility of pollen and spikelets; pollen scale bar is 50 μm; spikelet scale bar is 5 cm; FF indicates all fertile, PF indicates approximately 75% fertile pollen, and SS indicates approximately 50% sterile pollen. Detailed Implementation
[0038] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.
[0039] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.
[0040] Example 1: Obtaining the S19g-A7 gene
[0041] This invention uses genetic mapping analysis to identify a locus, S19, located on chromosome 3 of African rice that controls male sterility in hybrid rice species from Asia and Africa. The genomic structure of the S19 locus is as follows: Figure 1 As shown in the figure. Sequence analysis revealed significant structural variation between Asian and African rice at the S19 locus. African rice exhibits a 94kb specific fragment within the S19g allele, containing a specific gene named S19g-A7, indicated by a black background in the figure. ORF1 and ORF2 are genes present in both Asian and African rice, indicated by a white background in the figure. Figure 1 (Figure A).
[0042] The gene structure of S19g-A7 is as follows: Figure 1 As shown in Figure B, exons are represented by black squares, and gray rectangles represent UTR regions.
[0043] The nucleotide sequence of S19g-A7 is shown in SEQ ID NO.1. SEQ ID NO.1:
[0044] ATG TAA
[0045] The amino acid sequence of S19g-A7 is shown in SEQ ID NO.2. SEQ ID NO.2:
[0046] MQGLSRSTLRLCRRPTVGFFIGSAPPLAVARRATPLIAAPSDLWNPISRFAFGPWGMQRYFGVKSENKPCKRDCTNIGFKPISPMKEMCCGTIEHKQSNLIQRPIKDV TESNQKTTDVNVTDSNHTIDVDKVAWCEHICQHSSHRDGTIYKYKLYWKNNYQIDVTNREETRVEPKRYSMDTGCIPNPENCLYHLTCQMVQIFSLKLAKTTINSGPLQ LYGYIAARDLVDDMLNYVFNRSRDDPIIVQEESIIEMTGPKRGIALIPDVLFEFDMRIKNGDEEDDLQLIDGIIEFQEILLPEKPTTVRITGDYGDVDMCLANVSNGVE ATVEVAISEPDHGFDLSISCVHFMMEKSKEFHLFGGTIGESRQLRRFVMAVFLDTVMHLKFKVDQKGSNVVEHCCSFESKLHGCASHQIKLENASILVKVTWSPLIE.
[0047] Example 2: Construction of S19g-A7 functional complementary vector
[0048] The nucleotide sequence of S19g-A7 is shown in SEQ ID NO.1, and the protein sequence it encodes is shown in SEQ ID NO.2.
[0049] For the S19g-A7 sequence, a primer was designed from -2151 to -2130 bp upstream of the start codon (as shown in SEQ ID NO. 3), and a primer was designed from 3389 to 3411 bp downstream of the stop codon (as shown in SEQ ID NO. 4). These primers served as the primer pair for amplifying the complete S19g-A7 gene fragment. The complete S19g-A7 genome fragment was amplified using this primer pair and then ligated into the binary expression vector pCAMBIA1300 to obtain the vector p1300-S19g-A7. t .
[0050] The primer pairs for amplifying the complete S19g-A7 gene fragment are shown in SEQ ID NO. 3-4.
[0051] SEQ ID NO.3:
[0052] GATTACGAATTCGAGCTCGGTACCAGGGATTGGGTACTCAGGTCT;
[0053] SEQ ID NO.4:
[0054] GACTCTAGAGGATCCCCGGGTACCCCTCCATCGGATGTATCAGCTA. Example 3S19g-A7 t Identification of functional complementary transformants
[0055] The vector p1300-S19g-A7 constructed in Example 2 t Agrobacterium was introduced and transformed into the Asian rice variety 9522. Transgenic selection was performed using primers shown in SEQ ID NO.5-6, and transgenic strains carrying the S19g-A7 transgene were selected. t Positive S19g-A7 t Functionally complementary transformant plants (e.g.) Figure 2 (As shown), the primer pairs used are as shown in SEQ ID NO.5-6.
[0056] SEQ ID NO.5: ATTTGTGTACGCCCGACAGT;
[0057] SEQ ID NO. 6: GTGCTTGACATTGGGGAGTT.
[0058] Example 4S19g-A7 t Observation of pollen and spikelet fertility of functionally complementary transformants
[0059] For S19g-A7 t Functionally complementary transformant plant S19g-A7 t #15 and S19g-A7 t Pollen from multiple florets of #16 was stained with I2-KI solution to determine pollen fertility, and the results were observed as follows: Figure 3 As shown, S19g-A7 t #15 and S19g-A7 t The pollen of the #16 transformant is fully chromatic, indicating that the pollen fertility is fully fertile (FF).
[0060] For S19g-A7 t Functionally complementary transformant plant S19g-A7 t #15 and S19g-A7 t The fertility of spikelets in #16 was observed, and the seed setting rate of spikelets in both transgenic plants was fully fertile.
[0061] The results in summary indicate that the transgenic S19g-A7 does not affect the development of male gametes in the transformed plants.
[0062] Example 5F1-S19g-A7 t Phenotypic analysis and segregation analysis of genotype and phenotype in offspring
[0063] (a) Transgenic hybrid F1-S19g-A7 t Fertility test results
[0064] The F1 plants produced by crossing 9522 with SG72 (SG72×9522) were used as a normal Asian-African rice hybrid control.
[0065] The S19g-A7 obtained in Example 3 t The functionally complementary transformant was crossed with SG72 to obtain the transgenic hybrid F1-S19g-A7. t .
[0066] Interspecific hybrids of rice in Asia and Africa, F1 and F1-S19g-A7 t Pollen samples were stained with I2-KI solution (lighter staining with I2-KI indicated aborted pollen), and the fertility of spikelets was observed. F1-S19g-A7 t The pollen and spikelet fertility test results are as follows Figure 4 As shown, the results indicate that:
[0067] The pollen fertility of the F1 hybrid control from the Asian and African rice species exhibited a typical semi-sterile (SS, i.e., approximately 50% pollen sterility) phenotype, F1-S19g-A7. t The fertile pollen rate increased to about 75% (about 75% of pollen is fertile, PF), and the spikelets were fully fertile.
[0068] (ii) F1-S19g-A7 t Segregation analysis of genotype and phenotype in offspring
[0069] Further analysis of interspecific hybrids of rice in Asia and Africa, specifically F1 and F1-S19g-A7. t Genotypes and phenotypes in the self-crossed offspring (F2) were segregated and analyzed. The results are shown in Table 1.
[0070] The results showed that no S19s genotype plants were detected in the F2 population of the interspecific hybrid control of rice in Asia and Africa. The segregation ratio of the S19 genotype was significantly skewed from Mendel's free segregation ratio (1:2:1), indicating that the S19s pollen produced in the control F1 anthers was sterile.
[0071] For S19g-A7 cells carrying transgenic hemizygosity in a heterozygous S19gS19s genotype background tThe segregation of the S19 locus and S19g-A7 in the self-pollinated progeny of the plant. t The segregation of transgenes all conformed to the expected 2:3:1 ratio, indicating that the genotype was S19s / A7. t In pollen, S19g-A7 t This protects pollen survival, thus breaking the interspecific hybrid sterility barrier, therefore S19g-A7 t Transformation lines can be used as compatibility lines for interspecific hybrids of rice in Asia and Africa.
[0072] Table 1F1-S19g-A7 t Genotypic and phenotypic segregation analysis of self-crossed progeny populations
[0073]
[0074] Note: gg, gs, and ss represent genotypes S19gS19g, S19gS19s, and S19sS19s, respectively; 7 t 7 t This indicates the genetically modified S19g-A7 t homozygous, 7 t – indicates a transgenic hemizygote, -- indicates no transgene, NA indicates absence. ***, P<0.001 indicates a significant difference.
[0075] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.
Claims
1. A rice male gamete protection protein S19g-A7, characterized by, Its amino acid sequence is shown in SEQ ID NO.
2.
2. A rice male gamete protection gene S19g-A7, characterized in that, Its nucleotide sequence is the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.
2.
3. An agent for overcoming sterility of an intergeneric hybrid of rice, characterized by comprising a polynucleotide encoding a protein having an amino acid sequence represented by SEQ ID NO: 1 or a functional fragment thereof. Including the expression promoter of gene S19g-A7 as described in claim 2.
4. The application of the protein S19g-A7 of claim 1, the gene S19g-A7 of claim 2, or the reagent of claim 3 in overcoming interspecific hybrid sterility or improving the fertility of interspecific hybrids in rice.
5. The application of the protein S19g-A7 of claim 1, the gene S19g-A7 of claim 2, or the reagent of claim 3 in the creation of distant hybrid rice affinity materials.
6. The application of the protein S19g-A7 of claim 1, the gene S19g-A7 of claim 2, or the reagent of claim 3 in interspecific hybridization breeding of rice.
7. A method for creating a rice hybridizing line, characterized by, The S19g-A7 gene described in claim 2 is introduced into rice for functional complementation or overexpression.
8. The creation method of claim 7, wherein, The aforementioned functional complementarity or overexpression involves using the S19g-A7 self-promoter, constitutive promoter, or pollen overexpression promoter to drive the expression of the S19g-A7 coding sequence.
9. A method of constructing fertile hybrid rice by overcoming interspecific hybrid sterility, characterized by, The rice hybrid compatible line created by the method described in claim 7 or 8 is hybridized with African rice to produce a fertile interspecific hybrid of Asian and African rice.
10. The method of any of claims 7-9, wherein, The rice mentioned is Asian rice.