Esr1 gene for improving rice stigma exposure rate and application thereof

Abstract

The application belongs to the technical field of biological genes, and particularly relates to an ESR1 gene for improving the stigma exposure rate of rice and application thereof. The nucleotide sequence of the ESR1 gene is shown as SEQ ID NO. 1. The application clones a gene ESR1 for controlling the stigma exposure rate of rice from Echinochloa fruticulus, and the gene encodes a cysteine endopeptidase. The knockout strain of the ESR1 gene has a reduced stigma exposure rate and a smaller stigma angle; the complementary strain of the ESR1 gene has an improved stigma exposure rate and a larger stigma angle. It is proved that the ESR1 gene has the function of improving the stigma exposure rate of rice by increasing the stigma angle, and has important utilization value in promoting the genetic improvement of the stigma exposure characteristics of sterile lines and improving the seed production yield of hybrid rice.

Description

Technical Field

[0001] This invention belongs to the field of biogene technology, specifically relating to an ESR1 gene that improves the stigma exposure rate in rice and its application. Background Technology

[0002] Since its successful development in the 1970s, hybrid rice has made significant contributions to increasing global food production and reducing poverty. For many years, the production and utilization of hybrid rice have primarily relied on indica-indica or japonica-japonica hybridization to leverage heterosis between varieties. However, the development of indica-japonica subspecies hybrid rice and the utilization of greater heterosis are important directions for the current and future development of hybrid rice. The seed production processes of three-line hybrid rice and two-line hybrid rice both require outcrossing to obtain seeds. Therefore, improving the outcrossing seed setting rate is one of the core elements for promoting cost reduction and efficiency improvement in hybrid rice development. Research shows that the stigma exposure rate of rice is significantly positively correlated with the outcrossing seed setting rate; the pollination and seed setting rate of the female parent with exposed stigmas can account for more than 70% of the total seed setting rate, constituting a major part of seed production yield (Li Qingrong, 1985; Tian Dacheng et al., 1990). In addition, improving the stigma exposure rate of japonica sterile lines is one of the important goals of hybrid rice breeding in addressing the low seed production of intersubspecies hybrid rice of the "japonica-indica-sterile" type.

[0003] The stigma exfoliation rate of rice (Oryza sativa L.) is mainly controlled genetically, and several genes related to stigma exfoliation rate have been identified. From the perspective of rice pistil structure, stigma size, stigma angle, and style length are all components affecting stigma exfoliation rate. Currently cloned genes that help increase stigma exfoliation rate include OsSYL2 (Dang et al., 2020) and OsSYL3 (Dang et al., 2021), which control style length, and OsSPL5 (Li et al., 2025), which controls stigma size. The limited number of cloned genes restricts the application of molecular breeding. Wild rice is the ancestor of cultivated rice. After domestication, the reproductive method changed from cross-pollination in wild rice to self-pollination in cultivated rice, and the stigma exfoliation characteristic of wild rice gradually transformed into the stigma non-exfoliation or low exfoliation rate in cultivated rice. The natural outcrossing characteristics and high stigma exposure rate of wild rice are important materials for studying outcrossing and seed setting, and also excellent genetic resources for improving the stigma trait of male-sterile hybrid rice lines. Therefore, discovering and cloning genes with high stigma exposure rate in wild rice will provide important genetic resources for improving the outcrossing and seed setting rate of male-sterile lines and for hybrid rice breeding. Summary of the Invention

[0004] This invention aims to provide an ESR1 gene for improving stigma exposure rate in rice and its application. The invention cloned a gene, ESR1, from wild rice to control stigma exposure rate. This gene encodes a cysteine ​​endopeptidase. Knockout lines of the ESR1 gene showed decreased stigma exposure rate and a smaller stigma angle; complementary lines of the ESR1 gene showed increased stigma exposure rate and a larger stigma angle. This demonstrates that the ESR1 gene of this invention has the function of increasing stigma exposure rate in rice by increasing the stigma angle, and has important application value in promoting the genetic improvement of stigma exposure characteristics in sterile lines and increasing the yield of hybrid rice seeds.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: an ESR1 gene for improving the stigma exposure rate of rice, wherein the nucleotide sequence of the ESR1 gene is shown in SEQ ID NO.1.

[0006] As one embodiment of the ESR1 gene for improving the stigma exposure rate of rice according to the present invention, the CDS sequence of the ESR1 gene is shown in SEQ ID NO.2.

[0007] As one embodiment of the ESR1 gene for improving the stigma exposure rate of rice according to the present invention, the amino acid sequence encoded by the ESR1 gene is shown in SEQ ID NO.3.

[0008] This invention also claims protection for the application of the ESR1 gene in improving the stigma exposure rate in rice.

[0009] As one embodiment of the application of the ESR1 gene described in this invention in improving the stigma exposure rate of rice, the ESR1 gene increases the stigma exposure rate of rice by increasing the stigma angle.

[0010] As one embodiment of the application of the ESR1 gene in improving the stigma exposure rate of rice, the rice is cultivated rice (Oryza sativa).

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention screened a chromosome single-segment substitution line, SSSL29, with a high stigma exposure rate trait from a chromosome single-segment substitution line using wild rice (Oryza glumaepatula) as the donor and cultivated rice variety HJX74 as the recipient. SSSL29 was crossed and backcrossed with the recipient parent HJX74 to construct near-isogenic lines NIL-SG29 and NIL-HJX74 (hereinafter referred to as HJX74). Then, the gene ESR1, representing the high stigma exposure rate, was cloned using map-based cloning.

[0012] This invention constructs an ESR1 knockout vector and introduces it into NIL-SG29 via Agrobacterium-mediated transformation. The resulting knockout lines all exhibited reduced stigma exposure rates. A complementary vector derived from the ESR1 allele of wild rice was constructed, and the ESR1 gene and its promoter were introduced into HJX74 via Agrobacterium-mediated transformation. The complementary lines obtained showed significantly higher stigma exposure rates than HJX74. Furthermore, microscopic observation of mature stigmas in HJX74, NIL-SG29, knockout lines, and complementary lines revealed that the stigma angle of NIL-SG29 was significantly larger than that of HJX74, the stigma angle of the knockout lines was significantly smaller than that of NIL-SG29, and the stigma angle of the complementary lines was significantly larger than that of HJX74. This indicates that the stigma angle is the main factor leading to changes in stigma exposure rate. This invention demonstrates through relevant experiments that the ESR1 allele derived from wild rice is an excellent allele for improving stigma exposure rate in rice, effectively enhancing it. Attached Figure Description

[0013] Figure 1 This document presents partial flowering florets (showing stigma exposure) and statistical results of stigma exposure rates for HJX74 (a cultivated rice variety, Huajingxian 74) and SSSL29 (a single-segment chromosome substitution line using wild rice IRGC104387 as the donor and HJX74 as the recipient) during the flowering period in embodiments of the present invention. In the figures, a represents partial flowering florets of HJX74 and SSSL29, with arrows indicating exposed stigmas, and a scale bar of 1 cm; b is a bar chart of stigma exposure rates, with *** indicating significant differences at the P < 0.001 level.

[0014] Figure 2 This describes the map-based cloning process of the high stigma exsertion rate gene ESR1 in this embodiment of the invention. SER represents the stigma exsertion rate, and *** indicates a significant difference at the P < 0.001 level.

[0015] Figure 3 This diagram shows partial spikelet images (indicating stigma exposure) and stigma exposure rate data for the near-isogenic lines HJX74 and NIL-SG29 during the flowering period in this embodiment of the invention. In the diagram, a represents the stigma exposure phenotype of HJX74 and NIL-SG29, with the arrow indicating the exposed stigma, and the scale bar is 1 cm; b is a bar chart of stigma exposure rate, with *** indicating significant differences at the P < 0.001 level.

[0016] Figure 4The figures show the stigma morphology of HJX74 (a cultivated rice variety, Huajingxian 74) and NIL-SG29 (containing the high stigma exposure gene ESR1 from wild rice) in this embodiment of the invention. Here, 'a' represents the stigma morphology of mature spikelets of HJX74 and NIL-SG29, with a scale bar of 1000 μm; 'bg' represents the phenotypic values ​​of each part of the stigma. An independent samples t-test based on the mean (two-tailed) was used, with *** indicating significant differences at the P < 0.001 level, and 'ns' indicating no significant differences.

[0017] Figure 5 This shows the gene editing process of the ESR1 knockout mutant line in this embodiment of the invention.

[0018] Figure 6 This is a graph showing the stigma morphology and stigma exposure rate of NIL-SG29 and ESR1 gene knockout mutant lines in this embodiment of the invention. In the graph, a represents the stigma exposure phenotype of the parents and the knockout mutants, with the arrow indicating the exposed stigma and the scale bar being 1 cm; b is a bar chart of stigma exposure rate, with *** indicating significant differences at the P < 0.001 level.

[0019] Figure 7 The images show the stigma morphology of the NIL-SG29 and ESR1 gene knockout mutants in this embodiment of the invention. In the figures, a represents the morphology of the mature stigmas of the NIL-SG29 and ESR1 gene knockout mutants before pollination and fertilization, with a scale bar of 1000 μm; bg represents the phenotypic values ​​of each part of the stigma, analyzed using an independent samples t-test based on the mean (two-tailed). *** indicates a significant difference at the P < 0.001 level, and ns indicates no significant difference.

[0020] Figure 8 This is a partial floret diagram (showing stigma exposure) and stigma exposure rate data of the HJX74 and ESR1 complementary transgenic lines during the flowering period in this embodiment of the invention. In the diagram, 'a' represents the stigma exposure phenotype of the parent and the complementary transgenic line, with the arrow indicating the exposed stigma, and the scale bar is 1 cm; *** indicates a significant difference at the P < 0.001 level.

[0021] Figure 9 This image shows the stigma morphology of the HJX74 and ESR1 gene complementary transgenic lines in this embodiment of the invention. In the image, 'a' represents the mature stigma morphology of the HJX74 and ESR1 gene complementary transgenic lines before pollination and fertilization, with a scale bar of 1000 μm; 'bg' represents the phenotypic values ​​of each part of the stigma. An independent samples t-test based on the mean (two-tailed) was used, with *** indicating significant differences at the P < 0.001 level, and 'ns' indicating no significant differences. Detailed Implementation

[0022] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the examples and comparative examples are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0024] Example 1: Cloning of ESR1, a gene for high stigma exposure in wild rice. Our team screened a single-segment substitution line, SSSL29, with a high stigma exhumation rate from a previously constructed population of chromosome-single-segment substitution lines (Zhao et al, 2019) using wild rice (IRGC104387) as the donor and HJX74 (the cultivated rice variety Huajingxian 74) as the recipient. The stigma exhumation rate of HJX74 was 26.35%, while that of SSSL29 was 60.34%. Figure 1 The screening method for the single-segment substitution line SSSL29 can be found in the following reference: Hanwei Zhao(#), Lingling Sun(#), Tianyi Xiong(#), Zhangqiang Wang, Yu Liao, Tuo Zou, MingminZheng, Zhe Zhang, Xiaoping Pan, Ning He, Guiquan Zhang, Haitao Zhu, ZiqiangLiu, Ping He, Xuelin Fu(*). Genetic characterization of the chromosome single-segment substitution lines of O. glumaepatula and O. barthii and identification of QTLs for yield-related traits. Mol Breeding, 2019, 39: 51-69.

[0025] The SSSL29 and HJX74 were crossed to construct the F2 mapping population. Phenotypic linkage analysis was performed using QTL IciMapping software. A major QTL qPES1a was identified on chromosome 1 with a LOD value of 3.49 and a QTL phenotypic contribution rate of 16.90%.

[0026] To further narrow down the mapping region and expand the F2 population, markers were densified and exchanged plants were screened, resulting in a 24.3 kb physical region that maps qPES1a to markers M77 and M79. Referring to the Nipponbare genome, this region was predicted to contain three genes: ORF1, ORF2, and ORF3. However, the reference genomes of HJX74 and wild rice only contain ORF1 and ORF3. Figure 2 ).

[0027] During the localization process, a secondary single-fragment substitution line, sSSSL4, was screened from the exchanged lines. Its stigma exhumation rate reached 55.42%. The substitution fragment contained the high stigma exhumation rate gene ESR1 from wild rice, which was used as the near-isogenic line NILSG29 for subsequent studies. Figure 3 , Figure 4 ).

[0028] Example 2: Sequence of ESR1 gene for high stigma exposure rate in wild rice. 1. CDS sequence cloning of ESR1 (ORF1) Primers CF (5'-ATGGGGAGGGTTATTAGCAG-3') and CR (5'-TCACTGGGTTTCCTTGGCGC-3') were designed based on the HJX74 genome sequencing results. The CDS sequence of the ESR1 gene was amplified using NILSG29 cDNA as a template. The full length was 1116 bp.

[0029] The specific steps are as follows: (1) Add liquid nitrogen to the ceramic mortar after high temperature and high pressure sterilization, then put in the young spikes of NILSG29 and grind them thoroughly into powder. Dispense the powder into 1.5ml sterile enzyme-free centrifuge tubes, 200μl per tube.

[0030] (2) Total RNA was extracted from the young spikelets of NILSG29 using the MolPure® TRIeasy Plus Total RNA Kit from Yisheng Biotechnology Co., Ltd. The specific operation steps were performed according to the instructions.

[0031] (3) Using the total RNA from (2) as a template, the Hieff RNA from Yisheng Biotechnology Co., Ltd. was used to... ® The cDNA was synthesized by reverse transcription using the qPCR SYBRGreen Master Mix reverse transcription kit, and the reaction conditions were performed according to the kit instructions.

[0032] (4) Use the above primers and KOD-Plus-Neo enzyme to amplify the cDNA sequence.

[0033] (5) The amplification system in (4) above: 1.5µl template DNA, 1.5µl CF, 1.5µl CR, 5µl Buffer for KOD-Plus-Neo, 5µl 2 mM dNTPs, 3µl 25 mM MgSO4, 1.5µl DMSO, 1µl KOD-Plus-Neo, 30µl lddH2O.

[0034] (6) The amplification program in (4) above: 94℃ pre-denaturation for 2 min, 38 cycles (98℃ denaturation for 10 sec, 60℃ extension for 30 sec, 68℃ extension for 1 min) for a total of 38 cycles, 68℃ extension for 7 min.

[0035] (5) Electrophoresis detection: The size of the PCR product bands was observed by 1% agarose gel electrophoresis. PCR products with the correct band size were sent to the company for sequencing.

[0036] 2. Cloning of the nucleotide sequence of ESR1 (ORF1) Primers GF (5'-CATTCATTTCAAGGAGGCCAACATG-3') and GR (5'-AGCTATTCACAAGTCAAGCAGCAC-3') were designed based on the HJX74 genome sequencing results. Using these primers, a 4663 bp genome fragment was amplified from the genomic DNA of the near-isogenous line NILSG29. This genome fragment includes a 2926 bp promoter sequence upstream of the start codon ATG, a 1116 bp gene sequence between the start codon ATG and the stop codon TGA, and a 621 bp sequence downstream of the stop codon TGA.

[0037] The specific steps are as follows: (1) Extraction of total DNA from NILSG29 using the CTAB method: Take a tender leaf about 1 cm long and place it in a centrifuge tube. Add a clean steel bead and 500 µl of 1.5 x CTAB. Place the centrifuge tube in a grinder and grind twice at 50 Hz / min, 2 min each time, until thoroughly ground and homogeneous. Remove the steel bead. Incubate in a water bath at 65-75℃ for 20-30 min. Add 350 µl of phenol-chloroform and mix well. Centrifuge at 12,000 rpm for 10 min. Gently pipette 500 µl of the supernatant and transfer it to a new 1.5 ml centrifuge tube. Add 1 ml of pre-cooled anhydrous ethanol and place in a -20℃ freezer for 30 min to precipitate DNA. Centrifuge at 12,000 rpm for 15 min. Discard the supernatant, add 700 µl-1000 µl of 75% ethanol, and centrifuge at 12,000 rpm for 5 min. After centrifugation, discard the supernatant, invert the precipitate onto a spread-out paper towel to dry, and add 200µl of ddH2O after the precipitate becomes transparent. Store at 4℃.

[0038] (2) KOD-Plus-Neo PCR amplification system: 1.5µl template DNA, 1.5µl GF, 1.5µl GR, 5µl Buffer for KOD-Plus-Neo, 5µl 2 mM dNTPs, 3µl 25 mM MgSO4, 1.5µl DMSO, 1µl KOD-Plus-Neo, 30µl ddH2O.

[0039] (3) Amplification program: 94℃ pre-denaturation for 2 min, 38 cycles (98℃ denaturation for 10 sec, 60℃ extension for 30 sec, 68℃ extension for 3 min), 68℃ extension for 7 min.

[0040] (4) Electrophoresis detection: The size of the PCR product bands was observed by 1% agarose gel electrophoresis. PCR products with the correct band size were sent to the company for sequencing.

[0041] Example 3: Validation of ESR1 gene knockout mutant Construction of ESR1 gene knockout vector Following CRISPR / Cas9 editing technology, two target sites were designed in the coding region of the ESR1 gene: Target1: 5'-ACCGTTCGACGAGAGGGATTTGG-3' and Target2: 5'-GGCATCAACGCGATCCGGAC-3'. After PCR amplification, gDNA expression cassettes U6a-T1-gRNA and U6b-T2-gRNA containing the target sites were obtained. These cassettes were then ligated into the PYLCRISPR / Cas9Pubi-H plasmid using the restriction endonuclease BsaI and T4 DNA ligase to obtain the PYLCRISPR / Cas9Pubi-H recombinant vector containing the aforementioned target sequences.

[0042] Genetic transformation and acquisition of mutant plants The constructed PYLCRISPR / Cas9Pubi-H recombinant vector was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation. Then, the recombinant Agrobacterium tumefaciens strain was used to infect the callus tissue of the recipient material NIL-SG29. Through callus screening, differentiation, rooting, hardening-off, and transplanting, T0 generation knockout mutant plants were finally obtained.

[0043] Identification of knockout plants: Using leaf DNA from T0 generation mutant plants as templates, specific primers Hyg-F (5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and Hyg-R (5'-TCCGACCTGATGCAGCTCTCGGAG-3') for the hygromycin resistance gene Hyg on the knockout vector were used. At the same time, a pair of identification primers Cas9-F (5'-GTGTCATCTATGTTACTAGATC-3') and Cas9-R (5'-CGATGTAGGAGATCGATGCATG-3') were designed with reference to the PYLCRISPR / Cas9Pubi-H recombinant vector sequence to perform PCR amplification reaction to detect whether the transgenic rice contains the recombinant vector.

[0044] Edit target detection Self-pollinated seeds from T0 generation mutant plants were harvested and planted into T1 generation lines. DNA was extracted and amplified using the two primer pairs Hyg-F and Hyg-R, and Cas9-F and Cas9-R, respectively. Single plants without hygromycin marker and Cas9 protein were screened, and their DNA was then used as templates for PCR amplification using primers ESR1-KO-F (5'-CTGGAGGGTTCTTGCGGTGG-3') and ESR1-KO-R (5'-GTACTCGAACGCGTTCTCCAT-3'). The amplified products were sequenced to screen for ESR1 mutations. Using the above method, homozygous ESR1 mutants ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 were screened in the T2 generation.

[0045] In ESR1-KO#1, compared to the near-isogenic line NIL-SG29, the ESR1 gene has an insertion of one deoxyribonucleotide A between positions 524-525 in the nucleotide sequence of SEQ ID NO.2. In ESR1-KO#2, compared to the near-isogenic line NIL-SG29, the ESR1 gene has a deletion of 19 deoxyribonucleotides between positions 90-109 in the nucleotide sequence of SEQ ID NO.2 (the deleted deoxyribonucleotides are TTCGACGAGAGGGATTTGG). In ESR1-KO#3, compared to the near-isogenic line NIL-SG29, the ESR1 gene has a deletion of one deoxyribonucleotide G at position 103 in the nucleotide sequence of SEQ ID NO.2. Specific mutation sites are as follows... Figure 5 As shown.

[0046] The T1 generation homozygous mutant single plants were harvested and propagated by continuous self-pollination to obtain the T3 generation homozygous mutant lines.

[0047] Mutant phenotypic analysis In the afternoon during the peak flowering period of rice, select the main panicles in which about 1 / 2 to 2 / 3 of the spikelets have opened that day and count the stigma exposure rate.

[0048] Samples were taken from the field before 9:00 AM or after 3:00 PM during the peak flowering period of rice. For each plant, about one-third of the main panicle that had flowered that day was taken. Eight mature spikelets that were about to open were selected from each panicle for stigma dissection. The samples were photographed under a Leica M205FA stereomicroscope. Then, using Image-Pro Plus measurement software, the various parts of the stigma were measured, and the stigma angle and stigma size were calculated.

[0049] Results analysis: (1) The results of the investigation on the stigma exposure rate of NIL-SG29 and mutants are as follows: Figure 6 As shown.

[0050] From the appendix Figure 6 The results show that the column exposure rates of mutants ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 were 49.49±5.05%, 55.43±6.93%, and 55.82±3.94%, respectively, which were significantly lower than those of NIL-SG29.

[0051] (2) Phenotypic investigation was conducted on the stigma angle and stigma size of NIL-SG29 and the mutant. The corresponding experimental results are as follows: Figure 7 As shown.

[0052] from Figure 7 It can be seen that the stigma angles of mutants ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 61.61±3.94°, 52.38±3.62°, and 54.85±2.20°, respectively; the stigma brush lengths of mutants ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 1.25±0.01mm, 1.24±0.01mm, and 1.25±0.05mm, respectively; and the stigma non-brush lengths of mutants ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 0.52±0.01mm, 0.55±0.02mm, and 0.05mm, respectively. 0.54±0.02mm; the stigma widths of ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 0.44±0.01mm, 0.45±0.02mm, and 0.48±0.03mm, respectively; the stigma lengths of ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 1.77±0.01mm, 1.79±0.03mm, and 1.79±0.05mm, respectively; the styla lengths of ESR1-KO#1, ESR1-KO#2, and ESR1-KO#3 are 0.25±0.01mm, 0.25±0.01mm, and 0.23±0.01mm, respectively. Statistical analysis showed that the stigma angle of the ESR1 mutant was smaller than that of NIL-SG29; the stigma brush length, stigma non-brush length, stigma width, stigma length, and style length of the ESR1 mutant were not significantly different from those of NIL-SG29.

[0053] Example 4: Verification of ESR1 gene complementation 1. Construction of genetic complementation vectors Primers PO-F (5'-GAGCTCGGTACCCGGGGATCCCATTCATTTCAAGGAGGCCAACATG-3') and PO-R (5'-ACGACGGCCAGTGCCAAGCTTAGCTATTCACAAGTCAAGCAGCAC-3') were designed based on the ESR1 gene and its upstream and downstream sequences. Using these primers, a 4663 bp genomic fragment was amplified from the genomic DNA of the near-isogenic line NILSG29. This fragment included a 2926 bp promoter sequence upstream of the ESR1 gene start codon ATG, a 1116 bp sequence between the start codon ATG and the stop codon TGA, and a 621 bp sequence downstream of the stop codon TGA. The 4663 bp genomic fragment was used to replace a small segment between the BamHI and HindIII restriction sites in the pCAMBIA1300 vector, keeping other sequences unchanged, to obtain the ESR1 complementary vector, named pCAMBIA1300-ESR1.

[0054] 2. Genetic transformation and identification of complementary transgenic lines The constructed complementary vector pCAMBIA1300-ESR1 was transferred into Agrobacterium EHA105 via electroporation. Then, callus tissue of the recipient material HJX74 was infected with positive Agrobacterium. Through callus selection, differentiation, rooting, hardening-off, and transplanting, transgenic plants were finally obtained. Seeds from the T0 generation transgenic plants were harvested and used for further propagation to obtain the T3 generation transgenic lines.

[0055] Genomic DNA was extracted from T3 generation complementary plants and wild-type plants using the CTAB method. Specific primers Hyg-F (5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and Hyg-R (5'-TCCGACCTGATGCAGCTCTCGGAG-3') for the tag gene antihygromycin on the complementary vector, forward primer P1-F (5'-Ccaggctttacactttatgc-3') and ESR1 gene-specific reverse primer (ESR1-COR 5'-GAGTCATTAGGAGAATCACAGC-3') were used; and specific primers ESR1 gene-specific forward primer (ESR1-COF 5'-GGCGTTCCTATGACTTATGTGAG-3') and reverse primers on the complementary vector (P2-R) were used. PCR detection was performed using 5'-gcgattaagttgggtaacgc-3'; wild-type plant genomic DNA was used as a negative control, and plants that could simultaneously amplify the above three bands in complementary plants were considered positive.

[0056] 3. Phenotypic analysis of complementary transgenic lines The sampling and survey results are the same as in Example 3.

[0057] Results analysis: (1) The results of the investigation on the stigma exposure rate of HJX74 and complementary transgenic lines are as follows: Figure 8 As shown.

[0058] from Figure 8 The experimental data show that the stigma exposure rates of the complementary lines ESR1-CO#1, ESR1-CO#2 and ESR1-CO#3 were 36.71±5.93%, 35.93±7.38% and 36.17±5.76%, respectively, which were significantly greater than those of HJX74.

[0059] (2) Phenotypic investigation of HJX74 and complementary transgenic lines, including stigma angle and stigma size, and the corresponding experimental results are as follows: Figure 9 As shown.

[0060] from Figure 9 The experimental data show that the stigma angles of the complementary lines ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 75.50±3.19°, 77.54±8.06°, and 62.76±4.88°, respectively; the stigma brush lengths of the complementary lines ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 1.24±0.04mm, 1.22±0.01mm, and 1.23±0.01mm, respectively; and the non-brush lengths of the stigmas of ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 0.58±0.03mm, 0.56±0.01mm, and 0.56±0.01mm, respectively. m, 0.52±0.03mm; the stigma widths of ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 0.42±0.02mm, 0.43±0.01mm, and 0.43±0.01mm, respectively; the stigma lengths of ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 1.82±0.05mm, 1.78±0.01mm, and 1.75±0.04mm, respectively; the style lengths of ESR1-CO#1, ESR1-CO#2, and ESR1-CO#3 are 0.24±0.01mm, 0.24±0.01mm, and 0.24±0.01mm, respectively. Statistical analysis showed that the stigma angle of the ESR1 complementary lines was greater than that of HJX74; the stigma brush length, stigma non-brush length, stigma width, stigma length, and style length of the ESR1 complementary lines were not significantly different from those of HJX74.

[0061] In summary, as can be seen from the above examples, this invention clarifies that the superior allele of the high stigma exposure rate gene ESR1 is derived from the allele of wild rice with bark emergence, and has the effect of improving the stigma exposure rate of rice. It has important utilization value in promoting the genetic improvement of the stigma exposure characteristic of sterile lines and increasing the yield of hybrid rice seed production.

[0062] The nucleotide sequence of the rice high stigma exposure rate gene in this invention is shown in SEQ ID NO.1, and is as follows: ESR1 nucleotide sequence The CDS sequence of the rice gene with high stigma exposure rate is shown in SEQ ID NO.2. This sequence consists of 1116 bases, as follows: CDS (Oryza glumaepatula) The protein encoded by the gene regulating high stigma exposure rate in rice has the amino acid sequence shown in SEQ ID NO.3, as follows: AA MGRVISSWRVLAVVAALMAMAAVELCAAIPFDERDLESDEALWDLYERWQEHHHVPRHHGEKHRRFGAFKDNVRYIHEHNKRGGRGYRLRLNRFGDMGREEFRATFAGSHANDLRRDGLAAPPLPGFMYEGVRDLPRAVDWRRKGAVTGVKDQGKCGSCWAFSTVVSVEGINAIRTGRLVSLSEQE LIDCDTADNSGCQGGLMENAFEYIKHSGGITTESAYPYRAANGTCDAVRARRAPLVVIDGHQNVPANSEAALAKAVANQPVSVAIDAGDQSFQFYSDGVFAGDCGTDLDHGVAVVGYGETNDGTEYWIVKNSWGTAWGEGGYIRMQRDSGYDGGLCGIAMEASYPVKFSPNRVTPRRALGAKETQ* The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A kind ESR1 The application of genes in improving the stigma exposure rate of rice is characterized by, The ESR1 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The ESR1 The CDS sequence of the gene is shown in SEQ ID NO.2; The ESR1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

3.

2. The application as described in claim 1, characterized in that, The ESR1 The gene increases the stigma exposure rate in rice by increasing the stigma angle.

3. The application as described in claim 1, characterized in that, The rice mentioned is cultivated rice.

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