Application of brachyglossum oryza gene SBPL6 in improvement of stigma traits of rice sterile line

Abstract

The invention relates to the technical field of biological genes, in particular to clone of an oryza brachytonkinensis gene SBPL6 and application of the oryza brachytonkinensis gene SBPL6 to improvement of stigma traits of a rice male sterile line, in particular to application of the oryza brachytonkinensis gene SBPL6 to improvement of stigma traits of a sterile line, and improvement of the stigma traits of the sterile line is beneficial to improvement of outcrossing ability of the sterile line. The cDNA (complementary deoxyribonucleic acid) sequence of the gene SBPL6 is shown as SEQ ID NO.2. The wild brachyglossum gene SBPL6 can effectively increase the hair brush length and the stigma length of a rice stigma, so that the stigma exsertion rate is increased, and the outcrossing capability of a sterile line is favorably improved.

Description

Technical Field

[0001] The present invention relates to the field of biological gene technology, and particularly relates to the application of the short-tongue wild rice gene SBPL6 in improving the stigma traits of rice male sterile lines. Background Art

[0002] Rice (Oryza sativa) is one of the most important food crops in the world and is the staple food for billions of people globally. The successful research, development and promotion of hybrid rice is a huge breakthrough in the history of rice production in China and even the world, which can increase the rice yield by more than 15 - 20%. However, the low seed production yield and high cost of hybrids have restricted the popularization and application of hybrid rice. In production, hybrid rice seed production is achieved by the outcrossing of male sterile lines and restorer lines. Research shows that the outcrossing ability of male sterile lines is significantly positively correlated with stigma traits, including stigma exsertion, stigma size, and stigma vitality, etc. Among them, the stigma size of rice affects the stigma exsertion and hybrid seed production yield of rice. However, since cultivated rice (Oryza sativa) (usually called rice) is a self-pollinated crop with small stigmas and a low stigma exsertion rate, so far, few large stigma genes have been identified and cloned from cultivated rice, and their functional mechanisms also need to be further analyzed, which is difficult to meet the needs of breeding improvement of the outcrossing characteristics of male sterile lines of hybrid rice. As the ancestor of cultivated rice, wild rice generally shows the characteristics of large stigmas and high stigma exsertion rate, and has obvious outcrossing characteristics. Therefore, exploring and utilizing the beneficial genes of large stigmas in wild rice will provide important support for the innovation of breeding resources and variety cultivation of hybrid rice. Summary of the Invention

[0003] The purpose of the present invention is to avoid the deficiencies in the prior art and provide the application of the short-tongue wild rice (O. barthii) gene SBPL6 in improving the stigma traits of rice male sterile lines (hereinafter referred to as male sterile lines for short). The short-tongue wild rice gene SBPL6 can effectively increase the brush length and length of rice stigmas, thereby increasing the stigma exsertion rate, and then promoting the improvement of the outcrossing ability of male sterile lines.

[0004] To achieve the above purpose, the present invention provides the following technical solutions:

[0005] Provide the application of the short-tongue wild rice gene SBPL6 in improving the stigma traits of rice male sterile lines. The stigma exsertion rate of the improved male sterile lines is significantly increased, and stigma traits beneficial to the improvement of outcrossing performance are provided. The cDNA sequence of the gene SBPL6 is as shown in SEQ ID NO.2.

[0006] In some embodiments, the stigma exsertion rate of the male sterile lines includes increasing the brush length and length of rice stigmas.

[0007] In some embodiments, the method for obtaining the gene SBPL6 includes: using Oryza brachyantha as the donor and the cultivated rice variety Huajingxian 74 as the recipient, through hybridization and backcrossing, combined with molecular marker-assisted selection, to obtain a stable and homozygous Oryza brachyantha chromosome single segment substitution line library;

[0008] Among them, the chromosome single segment substitution line SB58 with significantly longer stigma brush length, wider stigma width, and longer stigma length than the recipient parent HJX74 was selected, and the chromosome single segment substitution line SB58 was used as the basic material for cloning the large stigma gene SBPL6.

[0009] Provided is the application of the gene SBPL6 of Oryza brachyantha in improving the stigma traits of male sterile lines, and the gDNA sequence of the gene SBPL6 is shown as SEQ ID NO.1.

[0010] Provided is the application of the gene SBPL6 of Oryza brachyantha in improving the stigma traits of male sterile lines, and the AA sequence of the gene SBPL6 is shown as SEQ ID NO.3.

[0011] The present invention provides the beneficial effects of the application of the gene SBPL6 of Oryza brachyantha in improving the stigma traits of rice male sterile lines:

[0012] The present invention cloned the gene SBPL6 that controls the stigma brush length of rice from the single segment substitution line material of Oryza brachyantha. This gene has a specific transcript in Oryza brachyantha different from that of cultivated rice. The CDS sequence corresponding to this transcript encodes a protein that regulates the elongation growth of rice stigma. The stigma brush length and stigma length of the overexpression lines of the SBPL6 gene become longer, and the stigma exsertion rate is significantly increased, effectively improving the outcrossing ability of male sterile lines. Description of the Drawings

[0013] Figure 1 Shows the plant and stigma morphological characteristics of HJX74 (cultivated rice variety Huajingxian 74) and SB58 (homozygous single segment substitution line SSSL carrying the SBPL6 gene).

[0014] Figure 2 Shows the stigma characteristics of the SBPL6 gene knockout mutants.

[0015] Figure 3 Shows the stigma exsertion rate of the SBPL6 gene knockout mutants.

[0016] Figure 4 Shows the SBPL6 expression levels in HJX74 and the SBPL6 gene overexpression materials.

[0017] Figure 5 Shows the stigma characteristics of the SBPL6 gene overexpression materials.

[0018] Figure 6 The stigma exsertion rate of the material with overexpression of the SBPL6 gene.

[0019] Figure 7 The stigma traits of the new maintainer line SD20.

[0020] Figure 8 The stigma traits of the new sterile line S20L. Specific implementation manners

[0021] The preferred embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the preferred embodiments of the present invention are shown in the drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0022] Next, the present invention will further explain how to specifically provide a large stigma gene of Oryza brachyantha, and how to use the gene to increase the stigma length and stigma exsertion rate of rice so as to improve the stigma traits of the sterile line, in combination with the accompanying drawings and specific embodiments.

[0023] Example 1 Cloning of the large stigma gene of Oryza brachyantha

[0024] 1. Using Oryza brachyantha (IRGC103591) as the donor and HJX74 as the recipient, through hybridization, continuous backcrossing, and combined with molecular marker-assisted selection, a stable and homozygous chromosome single segment substitution line library of Oryza brachyantha was obtained, and a chromosome single segment substitution line SB58 as shown in Figure 1 was selected, whose stigma brush length, stigma width, and stigma length were all significantly greater than those of the recipient parent HJX74. This was used as the basic material for cloning the large stigma gene.

[0025] 2. The large stigma gene carried by SB58 in Oryza brachyantha was cloned by the map-based cloning method. It has a specific transcript different from that of cultivated rice, and has the function of increasing the stigma and improving the stigma exsertion rate, and can be used to improve the sterile line, thereby increasing the seed production yield of hybrid rice.

[0026] Example 2 Obtaining the sequence of the large stigma gene of Oryza brachyantha

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

[0028] The specific steps are as follows:

[0029] (1) Extract total DNA of rice by CTAB method: Put about 1 cm long young leaves into a centrifuge tube, add clean small steel beads and 500 μL of 1.5x CTAB extraction buffer. Put the centrifuge tube into a grinding machine and grind it twice at a frequency of 50 Hz / min for 2 min each time until the leaves are fully and evenly ground. Suck out the steel beads. Incubate in a water bath at 65 - 75 °C for 20 - 30 min, then add 350 μL of phenol-chloroform and mix well. Put the centrifuge tube into a centrifuge and centrifuge at 12,000 rpm for 10 min. Gently pipette 500 μL of the supernatant into a new 1.5 mL centrifuge tube, add 1 mL of pre-cooled absolute ethanol, and place it in a -20 °C refrigerator for 30 min to precipitate DNA. Then put the centrifuge tube into a centrifuge and centrifuge at 12,000 rpm for 15 min. Discard the supernatant, add 700 - 1000 μL of 75% ethanol, put it into a centrifuge and centrifuge at 12,000 rpm for 5 min. After centrifugation, discard the supernatant, invert it on a spread paper towel to dry the precipitate, and add 200 μL of ddH 2 O to dissolve the DNA and store it in a 4 °C refrigerator.

[0030] (2) Prepare KOD-Plus-Neo PCR amplification system (50 μL): 1.5 μL of template DNA, 1.5 μL of Primer 1, 1.5 μL of Primer 2, 5 μL of Buffer for KOD-Plus-Neo, 5 μL of 2 mM dNTPs, 3 μL of 25 mM MgSO 4 、1.5 μL of DMSO, 1 μL of KOD-Plus-Neo, 30 μL of ddH 2 O.

[0031] (3) PCR amplification program: Pre-denature at 94 °C for 2 min, 38 cycles (denature at 98 °C for 10 sec, extend at 60 °C for 30 sec, extend at 68 °C for 2 min), and extend at 68 °C for 7 min.

[0032] (4) Electrophoresis detection: Detect the amplified product bands by 1% agarose gel electrophoresis, and send the amplified products with band sizes between 1000 bp - 2000 bp for sequencing by referring to DL2000 DNA Marker.

[0033] 2. Referring to 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’). Using the Total RNA Extraction Kit (TRleasy Plus Total RNA Kit) from Shanghai Yisheng Biotechnology Co., Ltd., total RNA was extracted from the young panicles of SB58, and the II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) premix from Shanghai Yisheng Biotechnology Co., Ltd. was used for reverse transcription to synthesize cDNA. The CDS sequence of the gene, with a full length of 504 bp (attached in the experimental data section), was amplified from the reverse-transcribed cDNA using the above primers PF2 and PR2.

[0034] The specific steps are as follows:

[0035] (1) Add liquid nitrogen to a ceramic mortar, then put in the sample, grind it into fine powder, aliquot 200 μL into each tube, add 1 mL LB Buffer A1, and mix vigorously by shaking.

[0036] (2) Extract the total RNA from the young panicles of SB58 according to the operation instructions of the Total RNA Extraction Kit from Shanghai Yisheng Biotechnology Co., Ltd.

[0037] (3) Using the total RNA in (2) as a template, the II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) premix from Shanghai Yisheng Biotechnology Co., Ltd. was used for reverse transcription to synthesize cDNA, and the reaction conditions were carried out according to the kit instructions.

[0038] (4) Using the above primers PF2, PR2 and KOD-Plus-Neo enzyme, PCR amplification was carried out with cDNA as a template. Pre-denaturation at 94 °C for 2 min, then denaturation at 98 °C for 10 sec, extension at 60 °C for 30 sec, extension at 68 °C for 1 min, for a total of 38 cycles, and extension at 68 °C for 7 min.

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

[0040] Example 3 Construction of gene knockout mutants

[0041] 1. Construction of CRISPR / Cas9 vector: The PYLCRISPR / Cas9Pubi-H vector was used in this invention. The Cas9 protein expression cassette was integrated into the binary vector. The multiple cloning site Bsa I for loading multiple sgRNA expression cassettes was located near the RB of the binary vector. The sgRNA expression cassette elements were set on the intermediate plasmid vector, which could be assembled by restriction enzyme digestion, ligation and PCR methods, and then assembled onto the binary vector by the Golden Gate cloning method.

[0042] 2. Obtaining mutant plants: The constructed knockout vector was transferred into Agrobacterium tumefaciens EHA105 by electroporation transformation. Then, the recombinant Agrobacterium tumefaciens carrying the knockout vector was used to infect the callus of the receptor material SB58. Through the processes of callus screening, differentiation and rooting, transgenic plants were finally obtained.

[0043] 3. Identification of positive plants: The amplification primers for the hygromycin resistance gene (Hyg): Hyg-F (5’-ACGGTGTCGTCCATCACAGTTTGCC-3’) and Hyg-R (5’-TCCGACCTGATGCAGCTCTCGGAG-3’) were used as a pair of identification primers. At the same time, referring to 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 the transgenic T 0 -generation plant leaf gDNA as a template, a PCR amplification reaction was carried out to detect whether the transgenic plants contained the recombinant vector.

[0044] 4. Detection of editing targets: Using the transgenic T 0 -generation rice leaf gDNA as a template, a forward primer KO-F (5’-GCACAGAGACAGAGAAGAGCTCT-3’) and a reverse primer KO-R (5’-GGAGGGTGTGGAGCCTGACGAT-3’) were designed respectively upstream and downstream of the target site, and a PCR amplification reaction was carried out. Then, it was compared with the gDNA sequence of SB58 to analyze the editing situation of the target site.

[0045] 5. Homozygous mutant phenotype analysis: (1) Samples were taken in the field before 9:00 am or after 3:00 pm during the full flowering stage of rice. For each plant, the main panicle with 1 / 3 of the flowers blooming on the same day was selected, and 8 mature florets about to open were selected from each panicle to dissect the stigmas, which were then photographed under a Leica M205FA stereomicroscope. Then, the Image-Pro Plus software was used to measure the length of the stigma brush, the width of the stigma, and the length of the non-brush part of the stigma, and the stigma size was calculated.

[0046] (2) After 3:00 pm during the full flowering stage of rice, suitable plants were selected in the field to investigate the stigma exsertion rate (for the main panicles of the selected individual plants, more than 1 / 2 to 2 / 3 of the florets had bloomed and closed on the same day), and the stigma exsertion rate was statistically analyzed.

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

[0048] As Figure 3 shown, the investigation results of the stigma exsertion rate of SB58 and the knockout mutant lines showed that the stigma exsertion 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 extremely significantly smaller than that of SB58 (33.30 ± 5.44%).

[0049] Example 4 Construction of gene overexpression materials

[0050] 1. Construction of overexpression vector p35S::SBPL6: Using the cDNA of SB58 as a template, the full-length sequence of the gene was amplified with primers respectively carrying Kpn I and BamHI restriction sites, and the amplified product was purified and recovered; similarly, pOX-35S vector plasmid was double-digested with Kpn I and BamH I 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 colonies were picked for colony PCR, positive strains were obtained, and the plasmid was extracted after successful sequencing.

[0051] 2. Obtaining of overexpression plants: The constructed overexpression vector p35S::SBPL6 was transferred into Agrobacterium tumefaciens EHA105 by electroporation, and then the callus of receptor material HJX74 was infected with Agrobacterium tumefaciens carrying the overexpression vector. Through the processes of callus screening, differentiation and rooting, transgenic plants were finally obtained.

[0052] 3. Identification of overexpression positive plants: Using the gDNA of transgenic T 0 -generation rice leaves as a template, the amplification primers Hyg-F (5’-ACGGTGTCGTCCATCACAGTTTGCC-3’) and Hyg-R (5’-TCCGACCTGATGCAGCTCTCGGAG-3’) of the hygromycin resistance gene (Hyg), a tag gene on the overexpression vector, were used as a pair of identification primers, the forward primer Ubi-seq-F (5’-GCCTTCATACGCTATTTATTTGC-3’) on the overexpression vector and the gene-specific reverse primer OE-R (5’-GGCAGCCTGCCTGCAATGAG-3’) were used as the second pair of identification primers, and 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 as the third pair of identification primers for PCR amplification respectively to detect whether the transgenic plants contained the recombinant vector. Referring to DL2000 DNA Marker, if the amplified product bands of the three pairs of primers were around 500bp, 270bp and 578bp respectively, it indicated that the transgenic plant contained the recombinant vector.

[0053] As Figure 4 shown, the expression levels of genes in overexpression plants were detected by qRT-PCR. Using the total RNA extraction kit of Shanghai Yisheng Biotechnology Co., Ltd. TRleasy Plus Total RNA Kit), the total RNA of overexpression plants and HJX74 was obtained, and using the company II 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) The corresponding cDNA was obtained using the qPCR SYBR Green Master Mix premix. Using the qPCR SYBR Green Master Mix quantitative reagent, with the Actin gene as the internal reference, a pair of primers RT-F (5’-CATCAAGTCCCATGGCGAAG-3’) and RT-R (5’-CGGAGATAGTTGAGCCACCGGAG-3’) were used for qRT-PCR to detect the expression level of the target gene. The results showed that the gene expression level of the overexpressing plants was significantly increased.

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

[0055] (3) Result analysis: As Figure 5 shown, the phenotypic investigation results showed that the lengths of the stigma brush 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 lengths of the non-brush parts of the stigma 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 widths of the stigma 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; the lengths of the stigma 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 lengths of the stigma brush of the overexpressing lines were all significantly greater than that of HJX74 (1.23 ± 0.08 mm); the lengths of the non-brush parts of the stigma of the overexpressing lines were all less than that of HJX74 (0.57 ± 0.04 mm), and among them, the lengths of the non-brush parts of the stigma of OE#2 and OE#3 were significantly less than that of HJX74; the widths of the stigma of the OE lines were all significantly greater than that of HJX74 (0.44 ± 0.05 mm); the total lengths of the stigma of the OE lines were all significantly greater than that of HJX74 (1.80 ± 0.12 mm).

[0056] As Figure 6 shown, the stigma exsertion rates of the overexpressing 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 greater than that of HJX74 (26.39 ± 4.64%).

[0057] Example 5 Application of Gene SBPL6 in the Improvement of Rice Sterile Lines

[0058] 1. Creation of the sterile line carrying the SBPL6 gene: SB58 carrying SBPL6 was crossed with the maintainer line Huazhen B (HZB) of the three-line hybrid rice and then self-crossed. Combining molecular marker-assisted selection, a new homozygous maintainer line SD20 into which the SBPL6 gene was transferred was obtained. Furthermore, by crossing the new maintainer line with the sterile line Huazhen A (HZA), a new sterile line S20L carrying the SBPL6 gene was cultivated.

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

[0060] (3) Result analysis: As Figure 7 shown, the phenotypic investigation results showed that the stigma brush length of the new maintainer line SD20 was 1.72 ± 0.02 mm, the stigma length was 2.27 ± 0.02 mm, and the stigma exsertion rate was 55.54 ± 2.02%. While the stigma brush length of the original maintainer line Huazhen B (HZB) was 1.10 ± 0.02 mm, the stigma length was 1.71 ± 0.04 mm, and the stigma exsertion rate was 12.39 ± 3.97%. Statistical analysis showed that the stigma brush length, stigma length, and stigma exsertion rate of the new maintainer line SD20 were all significantly greater than those of the original maintainer line Huazhen B (HZB).

[0061] As Figure 8 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 exsertion 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 exsertion rate was 28.31 ± 1.88%. Statistical analysis showed that the stigma brush length, stigma length, and stigma exsertion rate of the new sterile line S20L were all significantly greater than those of the original sterile line Huazhen A (HZA).

[0062] Among them,

[0063] SEQ ID NO.1 is as follows,

[0064] gDNA (Oryza barthii)

[0065]

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

[0067] cDNA (Oryza barthii)

[0068] CCAGATCGCTCAGTCTCACACCGCACAGAGACAGAGAAGAGCTCTAGAGAGAACGAGAGAGAGAGACAGAGAGAGAGAGGGAGAATGGGGAGGAGAGCTTGCTGCGCAAAGGAAGGGATGAAGAGAGGGGCATGGACGAGCAAGGAGGACGACATGCTTGCCTCCTACATCAAGTCCCATGGCGAAGGCAAGTGGCGCGAGGTCCCCCAACGAGCTGGTTTGAGGCGGTGCGGCAAGAGCTGCAGGCTCCGGTGGCTCAACTATCTCCGGCCTAACATCAAGCGCGGCAACATCGACGACGACGAGGAGGAGCTCATCGTCAGGCTCCACACCCTCCTCGGCAACAGGTGGTCTCTCATTGCAGGCAGGCCGCCGGCAGCCGCGGTGGCAGCACGCCGGACACCGCCAGAGCGACGGACGCGGCGTCGTCCAGCTCCGTCGTGCCGCCGGGCCAGCAGCAGCAGCCAGCCTCCCGCGCCGACACCGTGTGGGCGCCCAAGGCCGTGCGGTGCACGCGCGGGTTCTTCTTCCACGACCGTGAAACAGCGCCGCTCGCCGCGGCGGCGCCGGCGCCGGCAGGGGAATTAGGAGACGGCGATGACGCCGACTGCGACTACTACTGCAGCGGCAGCAGCTCGGCGGCGACGACGACGTCGTCGAGCTCGTTCCCGGCGGTCGTCGAGCCGTGCTTCTCCGCCGGCGACGACTGGATGGACGACGTGAGAGCCTTGGCGTCGTTTCTTGACACCGACGACGCCTGGAACTTGTGTGCGTGACACTAGTTCGTCGTCCGTACGTACCTACGTGTATGCATGTGTGCGTCTCATAGAATGAATATAAAATAATATTTTCTCAATAGCAGTTGCGGGTTTATGTATATAAAGTTTAATTTTTCCGTCTTATTTAAAATAAATTATATTTATTTTAAAAAATAAGATACACATAAAGCACTATTTATATTTTATTATCCAATAATAATAAAAAATACT

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

[0070] AA (Oryza barthii)

[0071] MGRRACCAKEGMKRGAWTSKEDDMLASYIKSHGEGKWREVPQRAGLRRCGKSCRL RWLNYLRPNIKRGNIDDDEEELIVRLHTLLGNRWSLIAGRPPAAAVAARRTPPERRTRRRP APSCRRASSSSQPPAPTPCGRPRPCGARAGSSSTTVKQRRSPRRRRRRQGN*

[0072] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Application of the short-tongued wild rice gene SBPL6 in improving the stigma trait of rice sterile lines. The stigma exposure rate of the improved rice sterile lines is significantly improved, providing a stigma trait that is beneficial to improving outcrossing performance. The cDNA sequence of the gene SBPL6 is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The improvement of the stigma trait of the rice sterile line includes increasing the stigma brush length, the stigma length, and improving the stigma exposure rate.

3. The use according to claim 1, characterized in that: The method for obtaining the gene SBPL6 includes: using short-tongued wild rice as a donor and cultivated rice variety Huajingxian 74 as a recipient, through hybridization and backcrossing, combined with molecular marker-assisted selection, to obtain a stable homozygous short-tongued wild rice chromosome single segment substitution line library; Among them, the chromosome single segment substitution line SB58 was selected, which showed that the stigma brush length, stigma width and stigma length were significantly greater than the recipient parent cultivated rice variety Huajingxian 74, and the chromosome single segment substitution line SB58 was used as the basic material for cloning the large stigma gene SBPL6.

4. Application of the short-tongued wild rice gene SBPL6 in improving the stigma trait of rice sterile lines. The improved stigma trait of rice sterile lines helps to improve outcrossing ability. The gDNA sequence of the gene SBPL6 is shown in SEQ ID NO.

1.

5. Application of the short-tongued wild rice gene SBPL6 in improving the stigma traits of rice sterile lines. The improved stigma traits of the rice sterile lines increase the stigma brush length and stigma length of the rice sterile lines, and increase the stigma exposure rate. The AA sequence of the gene SBPL6 is shown in SEQ ID NO.3.

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