Application of short tongue wild rice gene sbpl6 in improving stigma traits of rice sterile line

Abstract

The present application relates to the technical field of biological genes, and particularly relates to a short-tongue wild rice gene SBPL6 cloning and application thereof in improving stigma traits of a rice male sterile line, in particular, application of the short-tongue wild rice gene SBPL6 in improving stigma traits of the sterile line, and the improved stigma traits of the sterile line help to improve the outcrossing ability of the sterile line, the cDNA sequence of the gene SBPL6 is shown as SEQ ID NO. 2, the short-tongue wild rice gene SBPL6 can effectively increase the stigma brush length and the stigma length of rice, thereby improving the stigma exposure rate and helping to improve the outcrossing ability of the sterile line.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a gene of short-tongued wild rice. SBPL6 Application in improving the stigma traits of rice male-sterile lines. Background Technology

[0002] Rice ( Oryza sativa Rice is one of the world's most important food crops, serving as the staple food for billions of people globally. The successful research and promotion of hybrid rice represents a significant breakthrough in the history of rice production in my country and the world, potentially increasing rice yield by 15-20% or more. However, low yield and high cost of hybrid seed production have hindered its widespread application. In production, hybrid rice seed production is achieved through cross-pollination between sterile and restorer lines. Research shows a significant positive correlation between the cross-pollination ability of sterile lines and stigma traits, including stigma exposure, stigma size, and stigma vigor. Among these, stigma size affects stigma exposure and hybrid seed production yield. However, due to the limitations of cultivated rice (… Oryza sativa Rice (commonly known as paddy rice) is a self-pollinating crop with small stigmas and low stigma exposure rate. To date, very few genes with large stigmas have been identified and cloned from cultivated rice, and their functional mechanisms require further analysis, making it difficult to meet the needs of breeding improvement for the outcrossing and seed-setting characteristics of hybrid rice male-sterile lines. Wild rice, as the ancestor of cultivated rice, generally exhibits large stigmas and high stigma exposure rate, demonstrating significant outcrossing characteristics. Therefore, discovering and utilizing the beneficial genes with large stigmas in wild rice will provide important support for the innovation of hybrid rice breeding resources and variety development. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a short-tongued wild rice ( O. Oryza barthii )Gene SBPL6 Application in improving the stigma trait of rice male-sterile lines (hereinafter referred to as male-sterile lines). This short-tongued wild rice gene... SBPL6 It can effectively increase the length of the stigma brush and the length of the stigma in rice, thereby increasing the stigma exposure rate and promoting the cross-pollination ability of sterile lines.

[0004] To achieve the above objectives, the present invention provides the following technical solution:

[0005] Provides short-tongue wild rice genes SBPL6 Application in improving the stigma trait of rice male-sterile lines: The improved male-sterile lines showed a significant increase in stigma exposure rate, providing a stigma trait beneficial for improving outcrossing performance. The gene... SBPL6 The CDS sequence is shown in SEQ ID NO.2.

[0006] In some embodiments, the stigma exposure rate of the sterile line includes increasing the stigma brush length and stigma length of the rice.

[0007] In some embodiments, the gene SBPL6 The acquisition methods include: using wild rice with short tongue as the donor and cultivated rice variety Huajingxian 74 as the recipient, through hybridization and backcrossing, combined with molecular marker-assisted selection, to obtain a stable homozygous single-segment substitution line library of wild rice chromosomes with short tongue.

[0008] Among them, the chromosome single-segment substitution line SB58, which exhibits significantly larger stigma brush length, stigma width, and stigma length than the recipient parent HJX74, was selected. This chromosome single-segment substitution line SB58 was used as the cloning line for the large stigma gene. SBPL6 The basic materials.

[0009] Provides short-tongue wild rice genes SBPL6 Application of the gene in improving the stigma traits of sterile lines SBPL6 The gDNA sequence is shown in SEQ ID NO.1.

[0010] Provides short-tongue wild rice genes SBPL6 Application of the gene in improving the stigma traits of sterile lines SBPL6 The encoded amino acid sequence is shown in SEQ ID NO.3.

[0011] This invention provides a short-tongued wild rice gene. SBPL6 Beneficial effects of its application in improving the stigma traits of rice male-sterile lines:

[0012] This invention cloned the gene controlling the length of the stigma brush in rice from a single-segment substitution line of short-tongued wild rice. SBPL6 This gene has a specific transcript in wild rice that differs from that in cultivated rice. The CDS sequence corresponding to this transcript encodes a protein that regulates stigma elongation in rice. SBPL6 The stigma brush length and stigma length of the gene-overexpressing lines were both increased, and the stigma exposure rate was significantly improved, which effectively enhanced the crossbreeding ability of the sterile lines. Attached Figure Description

[0013] Figure 1 For HJX74 (cultivated rice variety Huajingxian 74) and SB58 (carrier rice variety) SBPL6 The plant and stigma morphology characteristics of the homozygous single-segment substitution line (SSSL) were analyzed. Specifically: a. plant type at maturity (scale bar: 10 cm); b. spike type at maturity (scale bar: 10 cm); c. stigma morphology of mature spikelets (scale bar: 1000 μm); d. phenotypic values ​​of each stigma component, analyzed using an independent samples t-test based on the mean (two-tailed). ** Indicates in P A difference of ≤ 0.01 is considered significant, and ns indicates that the difference is not significant.

[0014] Figure 2 for SBPL6 Stigma characteristics of gene knockout mutants. Specifically: a. Stigma morphology of mature rice spikelets, scale bar 1000 μm; b. Phenotypic values ​​of various stigma components, analyzed using an independent samples t-test based on the mean (two-tailed). ** Indicates in P A difference of ≤ 0.01 is considered significant, and ns indicates that the difference is not significant.

[0015] Figure 3 for SBPL6 Spectrum exposure rate of gene knockout mutants.

[0016] Figure 4 For HJX74 and SBPL6 In gene overexpression materials SBPL6 Level of expression.

[0017] Figure 5 for SBPL6 Stigma characteristics of gene overexpression materials. Specifically: a. stigma phenotype of mature rice spikelets, scale bar 1000 μm; b. phenotypic values ​​of various stigma components, analyzed using an independent samples t-test based on the mean (two-tailed). ** Indicates in P A difference of ≤ 0.01 is considered significant, and ns indicates that the difference is not significant.

[0018] Figure 6 for SBPL6 Stigma exposure rate of gene overexpression materials.

[0019] Figure 7 The stigma characteristics of the new retainer system SD20 were determined.

[0020] Figure 8 The stigmatic traits of the new sterile line S20L. Detailed Implementation

[0021] Preferred embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While preferred embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0022] The present invention will be further explained below in conjunction with the accompanying drawings and specific embodiments, on how to provide a gene for a large stigma in short-tongued wild rice, and how to use the gene to increase the length and exposure rate of rice stigmas, thereby improving the stigma trait of sterile lines.

[0023] Example 1: Cloning of the large stigma gene in short-tongued wild rice

[0024] 1. Using short-tongued wild rice (IRGC103591) as the donor and HJX74 as the recipient, a stable homozygous single-segment chromosome substitution line library was obtained through hybridization, repeated backcrossing, and marker-assisted selection. Lines exhibiting significantly larger stigma brush length, stigma width, and stigma length than the recipient parent HJX74 were selected. Figure 1 The image shows a single chromosome segment substitution line, SB58. This was used as the basic material for cloning the large stigma gene.

[0025] 2. The gene controlling large stigma in short-tongued wild rice carried by SB58 was cloned using map-based cloning. It has a specific transcript that is different from that in cultivated rice and has the function of increasing stigma size and stigma exposure rate. It can be used to improve sterile lines and thus increase the yield of hybrid rice seed production.

[0026] Example 2: Obtaining the gene sequence of the large stigma of short-tongued wild rice

[0027] 1. Based on the HJX74 genome sequencing results, a pair of specific primers were designed (forward primer PF1: 5'-CCCCTATGGATTCTATTACTCC-3' and reverse primer PR1: 5'-CGAGTACTTGTATACTCCCTCG-3'). Rice total DNA was extracted using the CTAB method, and the full-length gDNA sequence of the gene was amplified using the above primers, yielding a full-length sequence of 1585 bp (attached in the experimental data column).

[0028] The specific steps are as follows:

[0029] (1) Extraction of total DNA from rice 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 extraction buffer. Place the centrifuge tube in a grinder and grind it twice at a frequency of 50 Hz / min, 2 min each time, until the leaf is thoroughly ground and uniform. Remove the steel bead. Incubate in a water bath at 65-75℃ for 20-30 min, then add 350 µL of phenol-chloroform and mix well. Place the centrifuge tube in a centrifuge and 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-chilled anhydrous ethanol and place in a -20℃ freezer for 30 min to precipitate DNA. Place the centrifuge tube in a centrifuge again and centrifuge at 12,000 rpm for 15 min. Discard the supernatant, add 700-1000 µL of 75% ethanol, and centrifuge at 12,000 rpm for 5 min. After centrifugation, discard the supernatant, invert the pellet onto a spread paper towel to dry, and once the pellet is clear, add 200 µL of ddH2O to dissolve the DNA. Store at 4°C.

[0030] (2) Prepare the KOD-Plus-Neo PCR amplification system (50 µL): 1.5 µL template DNA, 1.5 µL Primer 1, 1.5 µL Primer 2, 5 µL Buffer for KOD-Plus-Neo, 5 µL 2 mM dNTPs, 3 µL 25 mM MgSO4, 1.5 µL DMSO, 1 µL KOD-Plus-Neo, 30 µL ddH2O.

[0031] (3) PCR amplification program: 94℃ pre-denaturation for 2 min, 38 cycles (98℃ denaturation for 10 sec, 60℃ extension for 30 sec, 68℃ extension for 2 min), 68℃ extension for 7 min.

[0032] (4) Electrophoresis detection: The amplification product bands were detected by 1% agarose gel electrophoresis. The amplification products with band sizes between 1000 bp and 2000 bp were sent to the company for sequencing, referring to DL2000 DNAMarker.

[0033] 2. Based on the HJX74 genome sequencing results, a pair of specific primers were designed (forward primer PF2: 5'-ATGGGGAGGAGAGCTTGCTGCGC-3' and reverse primer PR2: 5'-TCACGCACACAAGTTCCAGGCGT-3'). Total RNA extraction was performed using the MolPure kit from Shanghai Yisheng Biotechnology Co., Ltd. ® Total RNA was extracted from SB58 young spikelets using the TRleasy Plus Total RNA Kit, and cDNA was synthesized by reverse transcription using the Hifair® II 1st StrandcDNA Synthesis SuperMix for qPCR (gDNA digester plus) premix from Shanghai Yisheng Biotechnology Co., Ltd. The CDS sequence of the gene was amplified from the cDNA obtained by reverse transcription using the above primers PF2 and PR2. The full length was 504 bp (attached in the experimental data column).

[0034] The specific steps are as follows:

[0035] (1) Add liquid nitrogen to a ceramic mortar, then add the sample and grind it into a fine powder. Dispense the powder into 200 μL tubes, add 1 mL of LB Buffer A1, and shake vigorously to mix.

[0036] (2) Total RNA was extracted from the young spikelets of SB58 according to the instructions of the Total RNA Extraction Kit of Shanghai Yisheng Biotechnology Co., Ltd.

[0037] (3) Using the total RNA in (2) as a template, cDNA was synthesized by reverse transcription using the Hifair® II 1stStrand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) premixed solution from Shanghai Yisheng Biotechnology Co., Ltd. The reaction conditions were carried out according to the kit instructions.

[0038] (4) PCR amplification was performed using the above primers PF2, PR2 and KOD-Plus-Neo enzyme, with cDNA as a template. The pre-denaturation was performed at 94℃ for 2 min, followed by denaturation at 98℃ for 10 sec, extension at 60℃ for 30 sec, and extension at 68℃ for 1 min, for a total of 38 cycles, with a final extension at 68℃ for 7 min.

[0039] (5) Electrophoresis detection: The amplification products were detected by 1% agarose gel electrophoresis. The amplification products with band sizes between 500 bp and 750 bp were sent to the company for sequencing to obtain the gene CDS sequence, referring to the DL2000 DNA Marker.

[0040] Example 3: Construction of gene knockout mutants

[0041] 1. CRISPR / Cas9 Vector Construction: This invention uses the PYLCRISPR / Cas9Pubi-H vector to integrate the Cas9 protein expression cassette into a binary vector, serving as a multiple cloning site for loading multiple sgRNA expression cassettes. Bsa I is located near the RB position of the binary vector. The sgRNA expression cassette element is located on the intermediate plasmid vector. It can be assembled using enzyme digestion ligation and PCR methods, and then assembled onto the binary vector using the Golden Gate cloning method.

[0042] Obtaining mutant plants: The constructed knockout vector was transferred into Agrobacterium EHA105 by electroporation. Then, the recombinant Agrobacterium carrying the knockout vector was used to infect the callus tissue of the recipient material SB58. Through callus selection, differentiation and rooting processes, transgenic plants were finally obtained.

[0043] 3. Identification of positive plants: Hyg-F (5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and Hyg-R (5'-TCCGACCTGATGCAGCTCTCGGAG-3') were used as a pair of identification primers for the hygromycin resistance gene (Hyg). Simultaneously, referencing the recombinant vector sequence after the sgRNA expression cassette was assembled into the PYLCRTSPR / Cas9Pubi-H vector, another pair of identification primers, Cas9-F (5'-GTGTCATCTATGTTACTAGATC-3') and Cas9-R (5'-CGATGTAGGAGATCGATGCATG-3'), were designed. Using gDNA from the leaves of transgenic T0 generation plants as templates, PCR amplification was performed to detect whether the transgenic plants contained the recombinant vector.

[0044] 4. Detection of editing target sites: Using gDNA from transgenic T0 generation rice leaves as a template, a forward primer KO was designed upstream and downstream of the target site, respectively. - F (5'-GCACAGAGACAGAGAAGAGCTCT-3') and a reverse primer KO - R (5'-GGAGGGTGTGGAGCCTGACGAT-3') was used for PCR amplification, and then compared with the gDNA sequence of SB58 to analyze the editing status of the target site.

[0045] 5. Phenotypic analysis of homozygous mutants: (1) Samples were taken in the field before 9:00 am or after 3:00 pm during the peak flowering period of rice. One-third of the main panicle of each plant that had flowered that day was taken. Eight mature spikelets that were about to open were selected from each panicle and the stigmas were dissected. The samples were photographed under a Leica M205FA stereomicroscope. The length of the stigma brush, the width of the stigma, and the length of the stigma non-brush were measured using Image-Pro Plus software, and the size of the stigma was calculated.

[0046] (2) After 3:00 pm during the peak flowering period of rice, select suitable plants in the field to investigate the stigma exposure rate (the selected single plant has 1 / 2 to 2 / 3 or more of the spikelets on the day of the main panicle has finished blooming and the spikelets have closed), and count the stigma exposure rate.

[0047] (3) Results analysis: such as Figure 2As shown, the results of the stigma size investigation of SB58 and the knockout mutants indicate that the stigma brush lengths of the knockout mutant lines KO#1, KO#2, and KO#3 are 1.24±0.08 mm, 1.25±0.02 mm, and 1.24±0.10 mm, respectively; the non-brusque lengths of the stigmas of KO#1, KO#2, and KO#3 are 0.61±0.07 mm, 0.62±0.06 mm, and 0.61±0.10 mm, respectively; the stigma widths of KO#1, KO#2, and KO#3 are 0.53±0.05 mm, 0.53±0.03 mm, and 0.52±0.08 mm, respectively; and the stigma lengths of KO#1, KO#2, and KO#3 are 1.85±0.15 mm, 1.87±0.07 mm, and 1.85±0.19 mm, respectively. Statistical analysis showed that the stigma brush length of the knockout mutant lines was significantly smaller than that of SB58 (1.57±0.07 mm); the stigma non-brush length of the knockout mutant lines was significantly larger than that of SB58 (0.51±0.02 mm); the stigma width of the knockout mutant lines was significantly smaller than that of SB58 (0.59±0.04 mm); and the stigma length of the knockout mutant lines was significantly smaller than that of SB58 (2.08±0.07 mm).

[0048] like Figure 3 As shown, the results of the investigation on the stigma exposure rate of SB58 and the knockout mutant lines showed that the stigma exposure rates of the knockout mutant lines KO#1, KO#2 and KO#3 were 25.40±7.12%, 22.70±5.80% and 23.38±4.98%, respectively, which were significantly lower than those of SB58 (33.30±5.44%).

[0049] Example 4: Construction of Gene Overexpression Materials

[0050] 1. Construction of overexpression vector p35S::SBPL6: Using SB58 cDNA as a template, the full-length sequence of the gene was amplified using primers with Kpn I and BamHI restriction sites, respectively. The amplification product was purified and recovered. Similarly, the vector plasmid pOX-35S was double-digested with Kpn I and BamHI and purified and recovered. Using a one-step rapid cloning kit, the target gene was recombined with the vector. The ligation product was transformed into DH5α competent cells. Single clones were picked for colony PCR to obtain positive strains. After successful sequencing, the plasmid was extracted.

[0051] 2. Obtaining overexpression plants: The constructed overexpression vector p35S::SBPL6 was transformed into Agrobacterium EHA105 by electroporation. Then, the callus tissue of the recipient material HJX74 was infected with Agrobacterium carrying the overexpression vector. Through callus selection, differentiation and rooting processes, transgenic plants were finally obtained.

[0052] 3. Identification of positive overexpression plants: Using gDNA from transgenic T0 generation rice leaves as templates, a pair of primers for identifying the hygromycin resistance gene (Hyg) on ​​the overexpression vector was used: Hyg-F (5'-ACGGTGTCGTCCATCACAGTTTGCC-3') and Hyg-R (5'-TCCGACCTGATGCAGCTCTCGGAG-3'). A second pair of primers was used: the forward primer Ubi-seq-F (5'-GCCTTCATACGCTATTTATTTGC-3') and the gene-specific reverse primer OE-R (5'-GGCAGCCTGCCTGCAATGAG-3') on the overexpression vector. Finally, the gene-specific forward primer OE-F (5'-TCAGGCTCCACACCCTCCTCGG-3') and the reverse primer Pox-rev-seq-R (5'-GGCAACAGGATTCAATCTTAAG-3') on the overexpression vector were used. The third pair of primers was used for PCR amplification to detect whether the transgenic plant contained the recombinant vector. Referring to the DL2000 DNA Marker, if the amplification products of the three primer pairs were approximately 500 bp, 270 bp, and 578 bp, respectively, it indicated that the transgenic plant contained the recombinant vector.

[0053] like Figure 4 As shown, the expression level of the overexpressing gene in the plant was detected by qRT-PCR, and the total RNA extraction kit (MolPure) from Shanghai Yisheng Biotechnology Co., Ltd. was used. ® Total RNA was obtained from overexpressing plants and HJX74 using the TRleasy Plus Total RNA Kit, and then processed using the company's Hifair® II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus Hieff). ® The corresponding cDNA was obtained using the qPCR SYBR Green Master Mix premix solution. The corresponding cDNA was then quantified using the company's Hieff® qPCR SYBR Green Master Mix reagent. Actin The gene was used as an internal control. qRT-PCR was performed using a pair of primers, RT-F (5'-CATCAAGTCCCATGGCGAAG-3') and RT-R (5'-CGGAGATAGTTGAGCCACCGGAG-3'), to detect the expression level of the target gene. The results showed that the gene expression level was significantly increased in the overexpressing plants.

[0054] 4. Phenotypic analysis of overexpression lines: The sampling and investigation methods are the same as those in (1) and (2) of Example 3.

[0055] (3) Results analysis: such as Figure 5 As shown, the phenotypic results indicate that the stigma brush lengths of the overexpressing lines OE#1, OE#2, and OE#3 were 1.43±0.09 mm, 1.45±0.04 mm, and 1.44±0.11 mm, respectively; the non-brusqueeze lengths of the stigmas of OE#1, OE#2, and OE#3 were 0.55±0.08 mm, 0.53±0.05 mm, and 0.52±0.04 mm, respectively; the stigma widths of OE#1, OE#2, and OE#3 were 0.50±0.03 mm, 0.51±0.01 mm, and 0.52±0.02 mm, respectively; and the stigma lengths of OE#1, OE#2, and OE#3 were 1.97±0.16 mm, 1.98±0.06 mm, and 1.95±0.12 mm, respectively. Statistical analysis showed that the stigma brush length of the overexpressing lines was significantly greater than that of HJX74 (1.23±0.08 mm); the stigma non-brush length of the overexpressing lines was significantly less than that of HJX74 (0.57±0.04 mm), with the stigma non-brush length of OE#2 and OE#3 being significantly less than that of HJX74; the stigma width of the OE lines was significantly greater than that of HJX74 (0.44±0.05 mm); and the total stigma length of the OE lines was significantly greater than that of HJX74 (1.80±0.12 mm).

[0056] like Figure 6 As shown, the stigma exposure rates of the overexpression lines OE#1, OE#2, and OE#3 were 51.00±6.65%, 51.74±6.09%, and 43.89±7.54%, respectively, which were significantly higher than those of HJX74 (26.39±4.64%).

[0057] Example 5 Gene SBPL6 Application in the improvement of rice male-sterile lines

[0058] 1. Creation of a male-sterile line carrying the SBPL6 gene: SB58, carrying the SBPL6 gene, was crossed with the three-line hybrid rice maintainer line Huazhen B (HZB) and self-crossed. Combined with marker-assisted selection, a new homozygous maintainer line SD20 carrying the SBPL6 gene was obtained. Then, by crossing the new maintainer line with the male-sterile line Huazhen A (HZA), a new male-sterile line S20L carrying the SBPL6 gene was bred.

[0059] 2. Phenotypic investigation of rice stigma traits: The sampling and investigation methods are the same as those in (1) and (2) of Example 3.

[0060] (3) Results analysis: such as Figure 7As shown, the phenotypic results indicate that the stigma brush length of the new maintainer line SD20 is 1.72±0.02 mm, the stigma length is 2.27±0.02 mm, and the stigma exposure rate is 55.54±2.02%, while the stigma brush length of the original maintainer line Huazhen B (HZB) is 1.10±0.02 mm, the stigma length is 1.71±0.04 mm, and the stigma exposure rate is 12.39±3.97%. Statistical analysis shows that the stigma brush length, stigma length, and stigma exposure rate of the new maintainer line SD20 are significantly greater than those of the original maintainer line Huazhen B (HZB).

[0061] like Figure 8 As shown, the stigma brush length of the new sterile line S20L was 1.50±0.08 mm, the stigma length was 2.03±0.03 mm, and the stigma exposure rate was 46.30±3.26%, while the stigma brush length of the original sterile line Huazhen A (HZA) was 1.17±0.01 mm, the stigma length was 1.76±0.01 mm, and the stigma exposure rate was 28.31±1.88%. Statistical analysis showed that the stigma brush length, stigma length, and stigma exposure rate of the new sterile line S20L were significantly greater than those of the original sterile line Huazhen A (HZA).

[0062] in,

[0063] SEQ ID NO.1 is shown below.

[0064] gDNA ( Oryza barthii )

[0065]

[0066] SEQ ID NO. 2 is as follows:

[0067] CDS ( Oryza barthii )

[0068] ATGGGGAGGAGAGCTTGCTGCGCAAAGGAAGGGATGAAGAGAGGGGCATGGACGAGCAAGGAGGACGACATGCTTGCCTCCTACATCAAGTCCCATGGCGAAGGCAAGTGGCGCGAGGTCCCCCAACGAGCTGGTTTGAGGCGGTGCGGCAAGAGCTGCAGGCTCCGGTGGCTCAACTATCTCCGGCCTAACATCAAGCGCGGCAACATCGACGACGACGAGGAGGAGCTCATCGTCAGGCTCCACACCCTCCTCGGCAACAGGTGGTCTCTCATTGCAGGCAGGCCGCCGGCAGCCGCGGTGGCAGCACGCCGGACACCGCCAGAGCGACGGACGCGGCGTCGTCCAGCTCCGTCGTGCCGCCGGGCCAGCAGCAGCAGCCAGCCTCCCGCGCCGACACCGTGTGGGCGCCCAAGGCCGTGCGGTGCACGCGCGGGTTCTTCTTCCACGACCGTGAAACAGCGCCGCTCGCCGCGGCGGCGCCGGCGCCGGCAGGGGAATTAG

[0069] SEQ ID NO. 3 is as follows:

[0070] Amino acid sequence ( Oryza barthii )

[0071] MGRRACCAKEGMKRGAWTSKEDDMLASYIKSHGEGKWREVPQRAGLRRCGKSCRLRWLNYLRPNIKRGNIDDDEEELIVRLHTLLGNRWSLIAGRPPAAAVAARRTPPERRTRRRPAPSCRRASSSSQPPAPTPCGRPRPCGARAGSSSTTVKQRRSPRRRRRRQGN*

[0072] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. Short-tongued wild rice gene SBPL6 Its application in improving the stigma traits of rice male-sterile lines is characterized by... The gene SBPL6 The CDS sequence is shown in SEQ ID NO.2; The improved rice male-sterile line is characterized by increased stigma length and stigma length, thereby increasing the stigma exposure rate.

2. Short-tongued wild rice gene SBPL6 Application in improving the stigma traits of rice male-sterile lines: The improved stigma traits of the rice male-sterile lines help to enhance cross-pollination ability, characterized by... The gene SBPL6 The gDNA sequence is shown in SEQ ID NO.1; the improved rice male sterile line stigma traits are to increase the stigma brush length and stigma length, and improve the stigma exposure rate.

3. The application of the short-tongued wild rice gene SBPL6 in improving the stigma trait of rice male-sterile lines, characterized in that, The amino acid sequence encoded by the gene SBPL6 is shown in SEQ ID NO.3; The improved rice male-sterile line is characterized by increased stigma length and stigma length, thereby increasing the stigma exposure rate.

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