Rice stigma width regulation gene qSTB-8 and application of encoded protein thereof

Abstract

The invention discloses a rice stigma width regulation gene qSTB-8 and application of a coding protein thereof, the nucleotide sequence of the rice stigma width regulation gene qSTB-8 is shown as SEQ ID NO.1, and the application is selected from any one or more than two of the following applications: (1) application in regulation of rice stigma width; (2) application in breeding for improving the rice stigma exsertion rate; and (3) breeding for improving the rice outcrossing rate. According to the invention, a wide-stigma indica rice maintainer line Sichuan 345B and a narrow-stigma high-quality fragrant rice maintainer line Sichuan 106B are hybridized and assembled into a BC4F5 positioning group, then through a map-based cloning method, an SSR molecular marker is used for preliminary positioning, and finally qSTB-8 is finely positioned in a 11.2 kb section between a marker SG930 and a marker SG950. The functional analysis of the qSTB-8 gene is carried out, and the qSTB-8 gene is transferred into Sichuan 106B, so that the transgenic offspring shows the wide stigma character.

Description

Technical Field

[0001] The invention relates to a rice stigma width regulating gene, and in particular to a rice stigma width regulating gene qSTB-8 and application of a protein encoded by the gene. Background Art

[0002] Rice ( Oryza sativa.L ) As one of the important food crops, it is the staple food of more than half of the world's population. The poor rice yield will directly affect food security and social stability. At present, urbanization, salinization and desertification have caused a reduction in cultivated land, making food security issues more prominent, so how to increase rice production is particularly important. In the 1950s and 1970s, rice experienced two leaps in yield. The first was "dwarf breeding" by introducing dwarf genes, which greatly increased rice yields; the second was the use and promotion of hybrid vigor, which pushed rice yields to another new height. The yield of hybrid rice seed production is an important factor affecting its promotion and application. Rice is a self-pollinating plant, and its flower organs are not conducive to cross-pollination. Improving the cross-pollination rate of rice has become one of the important ways to increase the yield of hybrid rice breeding and seed production.

[0003] The current three-line and two-line breeding systems have low hybrid seed production yields and high seed production costs due to the poor outcrossing performance of the sterile lines, which greatly limits the large-scale promotion of hybrid rice. How to improve the outcrossing characteristics of the sterile lines and the maintainer lines, thereby increasing the outcrossing fruiting rate of their maternal parents, is one of the key technologies for high-yield breeding and seed production of hybrid rice. There are many factors that affect the outcrossing fruiting of sterile rice lines, which can be mainly divided into two aspects: flowering habits and floral traits. Flowering habits mainly include pollen shedding habits, flowering duration, panicle emergence speed, panicle extension degree, etc.; floral traits mainly include anther length, filament length, stigma length, stigma width, style length, stigma exposure rate, stigma vitality, etc.

[0004] The rice stigma is an organ that receives pollen. The exposure of the stigma can significantly increase the probability of cross-pollination. The length of the style, the length and width of the stigma determine the pollination capacity of the female parent, which directly affects the reproduction of the sterile rice line and the yield of hybrid rice seed production. The study of the stigma trait can provide a theoretical basis for the genetic improvement of this trait, which is of great significance for guiding the selection of sterile lines and improving seed production. However, in recent decades, there have been few reports on QTLs / genes that regulate the development of rice stigma. Due to the differences in the stigma control mechanism of different rice materials, it is necessary to construct a segregating genetic population to analyze the stigma-related QTL / genes of some important breeding materials, especially the hybrid rice parent materials that have been promoted and applied in production.

[0005] However, the current research on rice stigma traits is still lagging behind the exploration of other important traits such as rice quality and resistance, and there are few reports on the specific application of stigma width-related trait genes in rice breeding practice. Existing evidence shows that although some sterile lines show significant characteristics of high combining ability, good rice quality and strong resistance in rice production, their poor stigma traits lead to low cross-pollination fruiting rates, which limits their wide application in production practice. This situation highlights the importance of in-depth research on rice stigma traits, especially genes related to width traits, in order to promote the progress and development of rice male sterile line cross-breeding.

[0006] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0007] The purpose of the present invention is to provide a rice stigma width regulating gene qSTB-8 and application of its encoded protein. The gene qSTB-8 can participate in the regulation of rice stigma width.

[0008] In order to achieve the above object, the present invention provides an application of a rice stigma width regulating gene qSTB-8 or a protein encoded thereof, wherein the nucleotide sequence of the rice stigma width regulating gene qSTB-8 is shown in SEQ ID NO.1, and the application is selected from any one or more of the following: (1) Application in regulating rice stigma width; (2) Application in rice breeding to improve the stigma exsertion rate; (3) Application in rice breeding to improve the outcrossing rate.

[0009] The nucleotide sequence of the rice stigma width regulating gene qSTB-8, SEQ ID NO.1 is as follows:

[0010] Preferably, the rice stigma width regulating gene qSTB-8 is used to transform rice cells, and then the transformed rice cells are cultivated into plants.

[0011] Preferably, the rice stigma width regulatory gene qSTB-8 is used to transform rice cells, and pCAMBIA1300 is used as an empty vector to construct a recombinant vector pCAMBIA1300-qSTB-8 containing the rice stigma width regulatory gene qSTB-8.

[0012] Preferably, the rice stigma width regulating gene qSTB-8 is used to transform rice cells, Agrobacterium tumefaciens is used as a host cell, and the recombinant vector pCAMBIA1300-qSTB-8 is transferred into the host cell.

[0013] Preferably, the rice stigma width regulating gene qSTB-8 is used to transform rice cells, rice callus is infected and co-cultured in host cells containing the rice stigma width regulating gene qSTB-8, and transgenic seedlings are obtained through screening and differentiation.

[0014] Preferably, the amino acid sequence of the protein encoded by the rice stigma width regulating gene qSTB-8 is shown in SEQ ID NO.2, as shown below: MEWDLKMPPAASWELADELENSGGGGVPAAVSSSSAAVGGGVNAGGGGRQECSVDLKLGGLGEFGGGGAQPRVAVAGELAKGKGPAAAATGAAAAASSAPAKRPRGAAAGQQQ CPSCAVDGCKEDLSKCRDYHRRHKVCEAHSKTPLVVVSGREMRFCQQCSRFHLLQEFDEAKRSCRKRLDGHNRRRRRKPQPDPMNSASYLASQQGARFSPFATPRPEASWTGMIK TEESPYYTHHQIPLGISSRQQHFVGSTSDGGRRFPFLQEGEISFGNGAGAGGVPMDQAAAAAAASVCQPLLKTVAPPPPPHGGGGSGGGKMFSDGGLTQVLDSDCALSLLSAPA NSTAIDVGGGRVVVQPTEHIPIAQPLISGLQFGGGGGSSAWFAARPHHQAATGAAATAVVVSTAGFSCPVVESEQLNTVLSSNDNEMNYNGMFHVGGEGSSDGTSSSLPFSWQ.

[0015] The application of the rice stigma width regulating gene qSTB-8 and the protein encoded therein of the present invention has the following advantages: The invention firstly constructs a positioning population, and a BC4F5 positioning population is formed by hybridizing a wide stigma indica rice maintenance line Chuan 345B and a narrow stigma high-quality aromatic rice maintenance line Chuan 106B, and then the qSTB-8 locus is preliminarily positioned in a 9.6cM region between markers RM23444-RM447 on chromosome 8 by using a map-based cloning method using SSR molecular markers, and finally qSTB-8 is finely positioned in an 11.2 kb segment between markers SG930 and SG950. On this basis, the qSTB-8 gene function is analyzed, and it is transferred into Chuan 106B, and it is found that the transgenic offspring exhibits a wide stigma trait, which lays a foundation for breeding research on improving rice exsertion rate and outcrossing rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Comparative photos (a) and statistical results (b) of the stigma width of Sichuan 106B and Sichuan 345B rice in Experimental Example 1 of the present invention.

[0017] Figure 2 This is the precise location and cloning diagram of the qSTB-8 gene in Experimental Example 1 of the present invention.

[0018] Figure 3 This is a map of the pCAMBIA1300-qSTB-8 vector in Experimental Example 2 of the present invention.

[0019] Figure 4 The comparison photos (a) and statistical results (b) of the stigma width of Zhongchuan 106B and transgenic complementary T1 rice in Experimental Example 4 of the present invention. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0021] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0022] In the present invention, all features defined in the form of numerical ranges or percentage ranges, such as values, quantities, contents and concentrations, are only for brevity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be deemed to have included and specifically disclosed all possible secondary ranges and individual values ​​within the range (including integers and fractions).

[0023] The features mentioned in the present invention can be combined arbitrarily, as long as there is no contradiction in the combination of these features, all possible combinations should be considered as the scope of this specification. Each feature disclosed in the specification can be replaced by any alternative feature that can provide the same, equal or similar purpose. Therefore, unless otherwise specified, the disclosed features are only general examples of equal or similar features.

[0024] Experimental Example 1 Cloning of Rice Stigma Width Regulatory Gene qSTB-8 1. Rice material Chuan 106B is a long-grain, high-quality, fragrant rice maintenance line, and Chuan 345B is an indica rice maintenance line. Figure 1 As shown, the capital of Sichuan 106B is thin and narrow, while the capital of Sichuan 345B is thick and wide.

[0025] 2. Genetic analysis and positioning populations After the hybridization of Chuan 106B and Chuan 345B, backcrossing with Chuan 106B as the recurrent parent for 4 generations and selfing for 5 generations, the BC4F5 positioning population was obtained. About 1 gram of tender leaves of each plant was taken at the seedling stage to extract total plant DNA.

[0026] 3. Localization of qSTB-8 gene The 685 pairs of SSR primers evenly distributed on 12 pairs of rice chromosomes stored in our laboratory were used to screen the primers for Chuan 106B and Chuan 345B. Due to experimental needs, the primers of the three target segments were encrypted, and 361 pairs of primers were added for screening. The average physical distance of the encrypted primers was 0.17 cM.

[0027] Genomic DNA was extracted using the CTAB method. The specific steps are as follows:

[0028] (1) Weigh 0.1 g of rice leaves and grind them into powder using liquid nitrogen. Then add 700 µL of CTAB solution (containing 2% (m / V) CTAB, 100 mmol / L Tris-C1, 20 mmol / L EDTA, and 1.4 mol / L NaCl; pH 8.0) to prepare DNA extraction buffer. Incubate in a water bath at 65°C for 1 hour, mixing several times during the water bath. (2) Add 700 µL of chloroform solution and mix well. Centrifuge at 12,000 rpm for 10 minutes and transfer the supernatant to a new centrifuge tube.

[0029] (3) Add an equal volume of pre-cooled isopropanol to the supernatant obtained after centrifugation in the above step, mix well, and place at -20°C for more than 30 minutes. Centrifuge at 12,000 rpm for 10 minutes and pour out the supernatant;

[0030] (4) Wash the DNA precipitate obtained in the above step with an equal volume of 70% ethanol (500 μL). Centrifuge at 12,000 rpm for 10 minutes and pour out the supernatant. Add 500 μL 100% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 10 minutes, pour out the supernatant and collect the precipitate. Dry the washed DNA and dissolve it in 100 μL TE buffer or pure water;

[0031] (5) The concentration of the DNA sample obtained in the above steps was detected by UV spectrophotometry, and the integrity of the DNA was detected by 0.7% agarose gel electrophoresis.

[0032] The PCR reaction system used was a 25 µL system: 2 µL DNA template, 12.5 µL Premix Taq™, 1 µL each of forward and reverse primers (10 µmol / L), and 8.5 µL ddH2O.

[0033] The PCR amplification program was as follows: pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, 35 cycles, and finally extension at 72°C for 5 minutes. The PCR products were electrophoresed on a 4% agarose gel, and the gel was photographed and read on a gel imager after the electrophoresis.

[0034] The linkage analysis of qSTB-8 gene using the above-screened SSR primers revealed linkage at SSR markers RM23444-RM447 on chromosome 8. Using 31 recombinant plants isolated from the F2 population of BC4F5 backcrossed with Chuan 106B, the newly designed primers were used to detect the recombinant plants, and qSTB-8 was finally precisely located in the 11.2 kb segment between markers SG930 and SG950 (see Figure 2 ).

[0035] 4. Candidate gene sequencing analysis According to the sequence information provided by the Rice Genome Annotation Database (http: / / rice.plantbiology.msu.edu / ), the 11.2 kb segment contains only one annotated gene, LOC_Os08g41940. PCR cloning and sequencing analysis of the gene revealed that there were five polymorphic sites in the coding region of Chuan106B and Chuan345B, of which two were synonymous mutations, located in exons 1 and 3, respectively; two were missense mutations, both located in exon 3; and one was an insertion mutation, located in exon 3. According to the gene annotation information in the MSU database, the candidate gene qSTB-8 encodes a gene GW8 that regulates rice grain width.

[0036] Experimental Example 2 Construction of pCAMBIA1300-qSTB-8 plant overexpression vector 1. Specific amplification primer design Based on the whole genome sequence of rice variety Oryza sativa L cv. Nipponbare provided by NCBI, specific primers for amplifying the full-length sequence of qSTB-8 candidate gene were designed, and specific restriction sites were added at both ends of the specific primers according to the characteristics of the selected pCAMBIA1300 expression vector and the qSTB-8 candidate gene sequence (see Figure 3 ). The specific amplification primers designed are: an EcoRI restriction site (GAATTC) is added to the 5' end of the forward primer (qSTB-8-F), and a KpnI restriction site (GGTACC) is added to the 5' end of the reverse primer (qSTB-8-R). The primer sequences are as follows:

[0037] qSTB-8-F (SEQ ID NO.3): GGATCCCAAAAGCCGCAATCTCGA; qSTB-8-R (SEQ ID NO. 4): GGTACCTTTTTCATTATTGTACTT.

[0038] 2. qSTB-8 gene amplification Then, the genomic DNA of the rice variety Chuan 345B was extracted, and the genomic DNA of Chuan 345B was used as a template to amplify the full-length sequence of the qSTB-8 candidate gene totaling 4477bp using the primers designed above (qSTB-8-F and qSTB-8-R), including the full-length qSTB-8 genomic DNA and its upstream and downstream sequences.

[0039] The PCR reaction system (10 µL) contained: 5 µL of 2× Phanta Max buffer, 0.8 µL of dNTP Mix (2.5 mM), 1 µL of Primer-F (10 µM), 1 µL of Primer-R (10 µM), 0.2 µL of Phanta Max Super-Fidelity DNA Polymerase, 0.5 µL of template, and 1.5 µL of deionized water.

[0040] PCR reaction program: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 4.5 minutes, 30 cycles; and final extension at 72°C for another 5 minutes.

[0041] 3. Construction of recombinant vector pCAMBIA1300-qSTB-8 The target fragment amplified by PCR was recovered, and the pCAMBIA1300 empty vector was linearized by double restriction digestion with EcoRI and KpnI, and the pCAMBIA1300 backbone was recovered. Then, T4 ligase, the recovered qSTB-8 candidate gene fragment and the pCAMBIA1300 empty vector after restriction digestion were used to prepare the reaction. The specific reaction system included: 2 µL of 5× T4 ligase premix, 3.5 µL of inserted target fragment, 0.5 µL of linearized vector, 2 µL of T4 ligase, and 3 µL of ddH2O. The reaction was carried out in a ligator at 25°C for 2.5 hours.

[0042] Then, the competent cells of E. coli DH5a were transformed, and the positive clones were screened by colony PCR identification and sequencing, and finally the complementary expression vector pCAMBIA1300-qSTB-8 ( Figure 3 ) 4. Transfect the host The pCAMBIA1300-qSTB-8 expression vector was transformed into Agrobacterium tumefaciens EHA105 by electroporation.

[0043] Experimental Example 3: Transformation of rice plant overexpression vector pCAMBIA1300-qSTB-8 into rice plant Sichuan 106B 1. Inducing callus (1) Select Sichuan 106B rice seeds with intact grains and no mold spots, remove the seed coat, disinfect the seeds with 75% ethanol three times, 1 minute each time, and wash with sterile water. After the water is drained, add 10% sodium hypochlorite solution, vacuum for 8 minutes, and then place on a shaker at 180 rpm, 30°C, and shake for 15 minutes;

[0044] (2) Pour out the sodium hypochlorite solution in a clean bench, wash several times with sterile water, spread the seeds on a sterilized plate filled with filter paper to absorb the moisture, and use tweezers to inoculate the seeds on NBD medium (pay attention to aseptic operation), with the embryo facing down or touching the medium. Culture in the dark at 28°C for 21 days, 12 to 14 seeds per tissue culture bottle.

[0045] 2. Subculture Remove the radicle and plumule around the callus, dry the callus on clean filter paper, transfer to NBD subculture medium, and culture in the dark at 28 ℃ for 7 to 9 days.

[0046] 3. Cultivation of Agrobacterium EHA105 The Agrobacterium culture obtained in Experimental Example 2 was spread on a YEP resistant plate and cultured in the dark at 28°C for about 36 hours. A single clone was selected for expansion and culture. 600 When the OD value is 0.3-0.8, it can be used for infection. Centrifuge at 4000rpm for 8 minutes at room temperature and discard the upper bacterial liquid to collect the bacteria. Resuspend the bacteria with an appropriate amount of AAM-As medium to induce toxicity, and finally make the bacterial liquid OD 600 The value is about 0.6. Keep it away from light for half an hour and then use it for infection.

[0047] 4. Infection and co-cultivation (1) Select callus tissue with good growth and compact structure, immerse it in the infection solution, mix it thoroughly by inverting, and let it stand at room temperature for 30 minutes; (2) Pour off the infection solution, transfer the callus to a plate containing sterilized filter paper and blow dry thoroughly, then transfer to the NBD-As co-culture medium. Add a piece of sterilized filter paper on the medium to prevent Agrobacterium from growing wildly, and culture in the dark at 28°C for 3 days. (3) Transfer the callus to a centrifuge tube, gently invert and mix, and let it stand for 15 minutes, mixing every 5 minutes; (4) Pour out the bacterial solution and place the callus on sterile filter paper to air dry for more than 1.5 hours to ensure that the bacterial solution is absorbed dry. Then, inoculate it on the NBD-As co-culture medium and culture it in the dark at 20°C for 2 to 3 days.

[0048] 5. Sterilization (1) After 3 days of co-culture, transfer the callus tissue to a triangular flask and wash it with sterile water for more than 3 times until the liquid is relatively clear; (2) Pour out the sterile water, wash with sterile water containing 100 mg / L cephalosporin and 100 mg / L carbenicillin until the water is clear, and shake at 200 rpm at 28°C for 20 minutes, 3 to 4 times; (3) Pour the washed callus onto sterile filter paper and blow dry thoroughly, then transfer to the screening medium and culture in the dark at 28°C. The medium can be replaced every 15 days.

[0049] 6. Differentiation The newly grown calli were transferred to differentiation medium and cultured at 28°C with 16 hours of light.

[0050] 7. Rooting The differentiated transgenic seedlings (>1 cm in height) were stripped of excess callus tissue and the roots were cut off (leaving about 0.5 cm), then transferred to 1 / 2 MS medium for rooting and cultured at 28°C with 16 h of light.

[0051] 8. Hardening and transplanting After rooting is completed, you can remove the rooting medium, soak the seedlings in water for several days to harden them, and then transplant them into the soil to grow.

[0052] Experimental Example 4 Identification of stigma traits of qSTB-8 overexpressing rice lines The plants transformed in Experimental Example 3 were identified and continuously observed, and the stigma width trait was examined and recorded. Figure 4 Compared with the stigma width of Chuan 106B at the same period, the stigma width of the transgenic complemented T1 generation plants was significantly increased.

[0053] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. Application of rice stigma width regulating gene qSTB-8 or its encoded protein, characterized in that: The nucleotide sequence of the rice stigma width regulating gene qSTB-8 is shown in SEQ ID NO.1, and the application is selected from any one or more of the following: (1) Application in regulating rice stigma width; (2) Application in rice breeding to improve the stigma exsertion rate; (3) Application in rice breeding to improve the outcrossing rate.

2. The use according to claim 1, characterized in that: The rice stigma width regulating gene qSTB-8 is used to transform rice cells, and then the transformed rice cells are cultivated into plants.

3. The use according to claim 2, characterized in that: The rice stigma width regulatory gene qSTB-8 is used to transform rice cells, and pCAMBIA1300 is used as an empty vector to construct a recombinant vector pCAMBIA1300-qSTB-8 containing the rice stigma width regulatory gene qSTB-8.

4. The use according to claim 3, characterized in that: The rice stigma width regulating gene qSTB-8 is used to transform rice cells, Agrobacterium tumefaciens is used as a host cell, and the recombinant vector pCAMBIA1300-qSTB-8 is transferred into the host cell.

5. The use according to claim 4, characterized in that: The rice stigma width regulating gene qSTB-8 is used to transform rice cells, rice callus is infected and co-cultured in host cells containing the rice stigma width regulating gene qSTB-8, and transgenic seedlings are obtained through screening and differentiation.

6. The use according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the rice stigma width regulating gene qSTB-8 is shown in SEQ ID NO.2.

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