Rice stigma width regulation gene qstb-8 and application of encoded protein thereof
By cloning and applying the rice stigma width regulation gene qSTB-8, the problem of insufficient research on rice stigma width regulation has been solved, the cross-pollination seed setting rate has been improved, and the breeding progress of sterile lines and the yield of hybrid rice seed production have been promoted.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- CROP INST SICHUAN PROVINCE ACAD OF AGRI SCI
- Filing Date
- 2025-02-27
- Publication Date
- 2026-06-26
Smart Images
Figure CN119876245B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to rice stigma width regulatory genes, specifically to the application of a rice stigma width regulatory gene qSTB-8 and its encoded protein. Background Technology
[0002] Rice ( Oryza sativa.L As a crucial food crop, rice is the staple food for more than half of the world's population. Insufficient rice yields directly impact food security and social stability. Currently, urbanization, salinization, and desertification are causing a reduction in arable land, making food security issues even more prominent. Therefore, increasing rice production is of paramount importance. Rice experienced two significant yield increases in the 1950s and 1970s. The first was through "dwarfing breeding," which introduced dwarfing genes, drastically increasing rice yields. The second was the utilization and promotion of hybrid vigor, pushing rice yields to new heights. Hybrid rice seed production yield is a crucial factor in its widespread application. Furthermore, rice is a self-pollinating plant, and its floral organs are not conducive to cross-pollination. Therefore, improving the cross-pollination seed setting rate of rice has become one of the important ways to increase the yield of hybrid rice seed production.
[0003] The current three-line and two-line breeding systems suffer from low hybrid seed production and high costs due to the poor outcrossing performance of the male-sterile lines, which greatly limits the large-scale promotion of hybrid rice. Improving the outcrossing characteristics of male-sterile lines and maintainer lines, thereby increasing the seed setting rate of their maternal parents, is one of the key technologies for high-yield hybrid rice propagation. Many factors affect the seed setting of male-sterile rice outcrosses, mainly divided into two aspects: flowering habits and floral traits. Flowering habits mainly include pollen shedding habits, flowering duration, heading speed, and panicle elongation; floral traits mainly include anther length, filament length, stigma length, stigma width, style length, stigma exposure rate, and stigma vigor.
[0004] The stigma of rice is the organ that receives pollen. Exposed stigmas significantly increase the probability of cross-pollination. Traits such as style length, stigma length, and width determine the pollination capacity of the maternal parent, directly affecting the reproduction of rice sterile lines and the seed yield of hybrid rice. Research on stigma traits can provide a theoretical basis for the genetic improvement of this trait, and is of great significance for guiding the breeding of sterile lines and improving seed yield. However, in recent decades, reports on QTLs / genes regulating rice stigma development have remained relatively few. Since the stigma control mechanisms differ among different rice materials, it is necessary to construct segregating genetic populations to analyze stigma-related QTLs / genes in some important breeding materials, especially hybrid rice parental materials that have been widely used in production.
[0005] However, research on rice stigma traits lags behind the exploration of other important traits such as rice quality and resistance, and there are even fewer reports on the specific applications of genes related to stigma width in rice breeding practices. Existing evidence suggests that although some male-sterile lines exhibit significant characteristics such as high combining ability, excellent rice quality, and strong resistance in rice production, their poor stigma traits lead to low cross-pollination seed setting rates, thus limiting their widespread application in actual production. This situation highlights the importance of in-depth research on rice stigma traits, especially genes related to stigma width, to promote the progress and development of cross-pollination breeding of rice male-sterile lines.
[0006] It should be noted that the information disclosed in the background section above is only used to enhance the understanding of the background of this disclosure, and therefore may include information that does not constitute prior art known to those skilled in the art. Summary of the Invention
[0007] The purpose of this invention is to provide a rice stigma width regulating gene qSTB-8 and its encoded protein, which can participate in the regulation of rice stigma width.
[0008] To achieve the above objectives, the present invention provides the application of the rice stigma width regulating gene qSTB-8 or its encoded protein, 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:
[0009] (1) Application in regulating the width of rice stigmas;
[0010] (2) Application in breeding to improve the stigma exposure rate of rice;
[0011] (3) Application in breeding to improve the crossbreeding rate of rice.
[0012] The nucleotide sequence of the rice stigma width regulating gene qSTB-8, SEQ ID NO.1, is shown below:
[0013]
[0014] Preferably, rice cells are transformed using the rice stigma width regulating gene qSTB-8, and then the transformed rice cells are cultured into plants.
[0015] Preferably, rice cells are transformed with the rice stigma width regulating gene qSTB-8, and a recombinant vector pCAMBIA1300-qSTB-8 containing the rice stigma width regulating gene qSTB-8 is constructed using pCAMBIA1300 as an empty vector.
[0016] Preferably, rice cells are transformed using the rice stigma width regulating gene qSTB-8, with Agrobacterium rhizogenes as the host cell, and the recombinant vector pCAMBIA1300-qSTB-8 is transferred into the host cell.
[0017] Preferably, rice cells are transformed using the rice stigma width regulating gene qSTB-8, rice callus tissue 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.
[0018] 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 follows:
[0019] MEWDLKMPPAASWELADELENSGGGGVPAAVSSSSAAVGGGVNAGGGGRQECSVDLKLGGLGEFGGGGAQPRVAVAGELAKGKGPAAAATGAAAAASSAPAKRPRGAAAGQQQ CPSCAVDGCKEDLSKCRDYHRRHKVCEAHSKTPLVVVSGREMRFCQQCSRFHLLQEFDEAKRSCRKRLDGHNRRRRRKPQPDPMNSASYLASQQGARFSPFATPRPEASWTGMIK TEESPYYTHHQIPLGISSRQQHFVGSTSDGGRRFPFLQEGEISFGNGAGAGGVPMDQAAAAAAASVCQPLLKTVAPPPPPHGGGGSGGGKMFSDGGLTQVLDSDCALSLLSAPA NSTAIDVGGGRVVVQPTEHIPIAQPLISGLQFGGGGGSSAWFAARPHHQAATGAAATAVVVSTAGFSCPVVESEQLNTVLSSNDNEMNYNGMFHVGGEGSSDGTSSSLPFSWQ.
[0020] The application of the rice stigma width regulating gene qSTB-8 and its encoded protein of the present invention has the following advantages:
[0021] This invention first constructed a localization population, the BC4F5 population, by crossing the broad-stigma indica rice maintainer line Chuan 345B with the narrow-stigma high-quality fragrant rice maintainer line Chuan 106B. Then, using map-based cloning, the qSTB-8 locus was initially located on chromosome 8 within a 9.6 cM region between markers RM23444 and RM447 using SSR molecular markers. Finally, qSTB-8 was finely localized to an 11.2 kb segment between markers SG930 and SG950. Based on this, functional analysis of the qSTB-8 gene was performed, and its transformation into Chuan 106B revealed that the transgenic offspring exhibited the broad-stigma trait, laying the foundation for breeding research aimed at improving rice emergence rate and outcrossing rate. Attached Figure Description
[0022] Figure 1 Photograph (a) and statistical results (b) comparing the stigma widths of rice varieties Chuan 106B and Chuan 345B in Experimental Example 1 of this invention.
[0023] Figure 2 This is a fine localization and cloning diagram of the qSTB-8 gene in Experimental Example 1 of this invention.
[0024] Figure 3 This is a diagram of the pCAMBIA1300-qSTB-8 carrier used in Experimental Example 2 of this invention.
[0025] Figure 4 Photograph (a) and statistical results (b) comparing the stigma width of Chuan 106B and transgenic complementary T1 generation rice in Experiment Example 4 of this invention. Detailed Implementation
[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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 skilled in the art without creative effort are within the scope of protection of the present invention.
[0027] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0028] In this invention, all features defined in the form of numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are used only for simplicity and convenience. Accordingly, the description of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values (including integers and fractions) within those ranges.
[0029] The features mentioned in this invention can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification, provided that there is no contradiction in the combination of these features. Each feature disclosed in the specification can be replaced by any alternative feature that provides the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the disclosed features are merely general examples of equivalent or similar features.
[0030] Example 1: Cloning of the rice stigma width regulating gene qSTB-8
[0031] 1. Rice materials
[0032] Chuan 106B is a maintainer line for long-grain, high-quality fragrant rice, while Chuan 345B is a maintainer line for indica rice. For example... Figure 1 As shown, the column head of column 106B is narrow, while the column head of column 345B is thick and wide.
[0033] 2. Genetic analysis and mapping populations
[0034] The BC4F5 population was obtained by crossing Chuan 106B with Chuan 345B, followed by backcrossing four generations using Chuan 106B as the recurrent parent and then self-crossing five generations. Approximately 1 gram of tender leaves from each seedling was collected during the seedling stage to extract total plant DNA.
[0035] 3. Location of the qSTB-8 gene
[0036] We used 685 pairs of SSR primers, evenly distributed across the 12 pairs of rice chromosomes preserved in our laboratory, to perform initial primer screening for Chuan106B and Chuan345B. Due to experimental needs, we then encrypted the primers for three target regions, adding 361 additional primer pairs for further screening. The average physical distance between the encrypted primers was 0.17 cM.
[0037] Genomic DNA was extracted using the CTAB method. The specific steps are as follows:
[0038] (1) Weigh 0.1g of rice leaves and grind them into powder with liquid nitrogen. Then add 700 µL of CTAB solution (containing 2% (m / V) CTAB, 100mmol / L Tris-C1, 20mmol / L EDTA and 1.4mol / L NaCl; pH 8.0) to prepare DNA extraction buffer. Incubate at 65℃ for 1 hour, mixing multiple times during the water bath process.
[0039] (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;
[0040] (3) Add an equal volume of pre-cooled isopropanol to the supernatant obtained after centrifugation in the above steps, mix well, and place at -20℃ for more than 30 minutes. Centrifuge at 12,000 rpm for 10 minutes and pour off the supernatant;
[0041] (4) Wash the DNA precipitate obtained in the above steps with an equal volume of 500 μL of 70% ethanol. Centrifuge at 12,000 rpm for 10 minutes and discard the supernatant. Add 500 μL of 100% ethanol to wash the precipitate, centrifuge at 12,000 rpm for 10 minutes, discard the supernatant, and collect the precipitate. Air dry the washed DNA and dissolve it in 100 µL of TE buffer or pure water;
[0042] (5) The concentration of the DNA sample obtained in the above steps was detected by ultraviolet spectrophotometry, and the integrity of the DNA was detected by 0.7% agarose gel electrophoresis.
[0043] 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.
[0044] The PCR amplification program was as follows: pre-denaturation at 94℃ for 5 minutes; denaturation at 94℃ for 30 seconds, annealing at 55℃ for 30 seconds, extension at 72℃ for 30 seconds, for 35 cycles, with a final extension at 72℃ for 5 minutes. PCR products were subjected to 4% agarose gel electrophoresis. After electrophoresis, the gel was photographed and read using a gel imaging system.
[0045] Linkage analysis of the qSTB-8 gene using the selected SSR primers revealed linkage at SSR markers RM23444-RM447 on chromosome 8. Using 31 recombinant individuals isolated from the F2 population of the backcross between BC4F5 and Chuan 106B, newly designed primers were used to detect the recombinant individuals. qSTB-8 was ultimately precisely mapped to an 11.2 kb region between markers SG930 and SG950 (see [link to relevant documentation]). Figure 2 ).
[0046] 4. Candidate gene sequencing analysis
[0047] According to the sequence information provided by the Rice Genome Annotation Database (http: / / rice.plantbiology.msu.edu / ), this 11.2 kb region contains only one annotated gene, LOC_Os08g41940. PCR cloning and sequencing analysis of this gene revealed five polymorphic sites in the coding region of Chuan106B and Chuan345B. Two of these 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, also located in exon 3. Based on gene annotation information in the MSU database, this candidate gene qSTB-8 encodes GW8, a gene that regulates rice grain width.
[0048] Example 2: Construction of pCAMBIA1300-qSTB-8 plant overexpression vector
[0049] 1. Design of specific amplification primers
[0050] Based on the complete genome sequence of the rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided in NCBI, specific primers for amplifying the full-length sequence of the qSTB-8 candidate gene were designed. Specific restriction enzyme sites were added to both ends of the specific primers according to the characteristics of the selected pCAMBIA1300 expression vector and the qSTB-8 candidate gene sequence (see [link to documentation]). Figure 3 The designed specific amplification primers are as follows: the forward primer (qSTB-8-F) has an EcoRI restriction site (GAATTC) added to the 5' end, and the reverse primer (qSTB-8-R) has a KpnI restriction site (GGTACC) added to the 5' end. The primer sequences are as follows:
[0051] qSTB-8-F (SEQ ID NO.3): GGATCCCAAAAGCCGCAATCTCGA;
[0052] qSTB-8-R (SEQ ID NO. 4): GGTACCTTTTTCATTATTGTACTT.
[0053] 2. qSTB-8 gene amplification
[0054] Then, genomic DNA was extracted from the rice variety Chuan 345B, and using Chuan 345B genomic DNA as a template, the full-length sequence of the qSTB-8 candidate gene, totaling 4477 bp, was amplified using the primers (qSTB-8-F and qSTB-8-R) designed above: including the full-length qSTB-8 genomic DNA and its upstream and downstream sequences.
[0055] The PCR reaction system (10 µL) contains: 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.
[0056] PCR reaction procedure: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 65℃ annealing for 15 seconds, 72℃ extension for 4.5 minutes, 30 cycles; final extension at 72℃ for 5 minutes.
[0057] 3. Construct the recombinant vector pCAMBIA1300-qSTB-8
[0058] The target fragment amplified by PCR was recovered. Simultaneously, the pCAMBIA1300 empty vector was linearized by double digestion with EcoRI and KpnI, and the pCAMBIA1300 backbone was recovered. Then, the reaction was prepared using T4 ligase, the recovered qSTB-8 candidate gene fragment, and the digested pCAMBIA1300 empty vector. The specific reaction system contained: 2 µL of 5× T4 ligase premix, 3.5 µL of the target fragment, 0.5 µL of linearized vector, 2 µL of T4 ligase, and 3 µL of ddH2O. The mixture was ligated in a ligator at 25°C for 2.5 hours.
[0059] Then, the cells were transformed into E. coli DH5α competent cells. Positive clones were screened by colony PCR and sequenced to obtain the qSTB-8 complementary expression vector pCAMBIA1300-qSTB-8. Figure 3 )
[0060] 4. Transfection of the host
[0061] The pCAMBIA1300-qSTB-8 expression vector was transformed into Agrobacterium tumefaciens EHA105 using electroporation.
[0062] Experiment 3: The pCAMBIA1300-qSTB-8 plant overexpression vector was transformed into rice chuan106B.
[0063] 1. Inducing callus tissue
[0064] (1) Select intact, mold-free Chuan 106B rice seeds, remove the seed coat, disinfect the seeds three times with 75% ethanol for 1 minute each time, and wash them with sterile water. After draining the water, add 10% sodium hypochlorite solution, vacuum for 8 minutes, and then place them on a shaker at 180 rpm and 30°C for 15 minutes.
[0065] (2) Pour off the sodium hypochlorite solution in the clean bench, wash several times with sterile water, spread it evenly in a pre-sterilized petri dish containing filter paper to absorb the water, and use tweezers to inoculate the seeds onto NBD medium (note the aseptic operation), with the embryo facing down or in contact with the medium, and incubate in the dark at 28 ℃ for 21 days, 12~14 seeds / tissue culture bottle.
[0066] 2. Subgeneration
[0067] Remove the radicles and plumules around the callus, let the callus dry on clean filter paper, transfer it to NBD subculture medium, and incubate in the dark at 28 ℃ for 7-9 days.
[0068] 3. Culture of Agrobacterium EHA105
[0069] The correctly cloned Agrobacterium culture obtained in Experiment 2 was plated on YEP-resistant plates and incubated in the dark at 28 °C for approximately 36 hours. Single colonies were picked and expanded for further culture until the Agrobacterium culture reached OD500. 600 A concentration of 0.3–0.8 is suitable for infection. After centrifugation at 4000 rpm for 8 minutes at room temperature, discard the supernatant and collect the bacterial cells. Resuspend the bacterial cells in an appropriate amount of AAM-As medium to induce virulence, ultimately achieving an OD value of [missing value]. 600 The value is around 0.6. After being kept in the dark for half an hour, it can be used for inoculation.
[0070] 4. Infection and Co-cultivation
[0071] (1) Select callus tissue with good growth and tight structure, soak it in the infection solution, mix thoroughly by inverting, and let it stand at room temperature for 30 minutes.
[0072] (2) Discard the infection solution, transfer the callus tissue to a petri dish containing sterile filter paper and dry it thoroughly, then transfer it to NBD-As co-culture medium. Add a sterile filter paper to the medium to prevent Agrobacterium from growing wildly, and incubate in the dark at 28°C for 3 days.
[0073] (3) Transfer the callus to a centrifuge tube, gently invert and mix, let stand for 15 minutes, and mix once every 5 minutes during this period;
[0074] (4) Pour out the bacterial solution, place the callus on sterile filter paper and blow dry for about 1.5 hours or more to ensure that the bacterial solution is dried, then transfer it to NBD-As co-culture medium and incubate in the dark at 20 ℃ for 2-3 days.
[0075] 5. Sterilization
[0076] (1) After co-culturing for 3 days, the callus tissue was transferred to an Erlenmeyer flask and washed with sterile water more than 3 times until the liquid was relatively clear.
[0077] (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, shake at 28°C and 200 rpm for 20 minutes, 3-4 times;
[0078] (3) After washing the callus tissue, pour it onto sterile filter paper and blow it dry thoroughly, then transfer it to the screening medium and incubate it in the dark at 28°C. The medium can be changed every 15 days.
[0079] 6. Differentiation
[0080] The newly grown callus after screening was transferred to differentiation medium and cultured at 28 °C under 16 hours of light.
[0081] 7. Rooting
[0082] 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). They were then transferred to 1 / 2 MS medium to root and cultured at 28 ℃ under 16 hours of light.
[0083] 8. Hardening off seedlings and transplanting
[0084] After rooting is complete, the rooting medium can be removed, and the seedlings can be soaked in water for several days to harden off before being transplanted into the soil to grow.
[0085] Experimental Example 4: Identification of stigma traits in qSTB-8 overexpressing rice lines
[0086] The plants transformed in Experiment 3 were identified and continuously observed, and the stigma width trait was investigated and recorded. See [link to relevant documentation]. Figure 4 Compared with the stigma width of Chuan 106B at the same time, the stigma width of the transgenic complementary T1 generation plants was significantly increased.
[0087] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.
Claims
1. The application of the rice stigma width regulating gene qSTB-8 or its encoded protein in increasing rice stigma width, characterized in that, The nucleotide sequence of the rice stigma width regulating gene qSTB-8 is shown in SEQ ID NO.
1.
2. The application according to claim 1, characterized in that, Rice cells were transformed using the rice stigma width regulating gene qSTB-8, and the transformed rice cells were then cultured into plants.
3. The application according to claim 2, characterized in that, Rice cells were transformed with the rice stigma width regulating gene qSTB-8, and a recombinant vector pCAMBIA1300-qSTB-8 containing the rice stigma width regulating gene qSTB-8 was constructed using pCAMBIA1300 as an empty vector.
4. The application according to claim 3, characterized in that, Rice cells were transformed using the rice stigma width regulating gene qSTB-8, and Agrobacterium rhizogenes was used as the host cell. The recombinant vector pCAMBIA1300-qSTB-8 was transferred into the host cell.
5. The application according to claim 4, characterized in that, Rice cells were transformed using the rice stigma width regulating gene qSTB-8. Rice callus tissue was then infected and co-cultured in host cells containing the rice stigma width regulating gene qSTB-8. Transgenic seedlings were obtained through screening and differentiation.
6. The application 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.