Application of OsMYB84 gene in regulation and control of rice seed size and drought resistance

Abstract

Through research, the invention provides application of the OsMYB84 gene in regulation and control of rice seed size and drought resistance, the coding sequence of the OsMYB84 gene is SEQ ID NO: 1, and the amino acid sequence of the OsMYB84 gene is SEQ ID NO: 2.

Description

Technical Field

[0001] This invention relates to the field of rice gene improvement, and in particular to the application of the OsMYB84 gene in regulating rice seed size and drought resistance. Background Technology

[0002] Rice is the staple food for more than half of the world's population, and its stable and high yields are crucial for ensuring food security. However, rice production is often constrained by abiotic stresses, among which drought is one of the most severe factors leading to yield losses.

[0003] In crop breeding, there is often a trade-off between yield and stress resistance (such as drought resistance), a phenomenon known as the "trade-off" effect. Traditional breeding methods, while attempting to increase rice yield, often result in a weakening of stress resistance traits such as drought resistance; conversely, focusing on enhancing drought resistance may limit yield potential. This negative genetic correlation makes it difficult to cultivate breakthrough varieties that synergistically enhance both high yield and high stress resistance through conventional hybridization and selection methods. Although several genes that separately regulate yield or drought resistance have been identified in current technologies, key genetic targets that can simultaneously break this trade-off and exert a synergistic positive regulatory effect on both traits are still rarely reported. This has become a major technical bottleneck in current high-yield and drought-resistant synergistic breeding.

[0004] Therefore, identifying and verifying key genes that can simultaneously and positively regulate rice yield-related traits (such as grain size and thousand-grain weight) and drought resistance, and elucidating their mechanisms of action, has significant theoretical value and application prospects for developing new molecular breeding strategies and cultivating new rice varieties with synergistic improvement in yield and drought resistance. This patented technology, through targeted mutation of the MYB84 gene (gene number: LOC_Os08g04840), discovered that its mutant (myb84) significantly increases rice seed length, grain width, and thousand-grain weight, while simultaneously enhancing drought resistance; while overexpression of MYB84 leads to the opposite phenotype, proving that this gene is an important target for simultaneously improving rice yield and drought resistance. This discovery reveals for the first time the dual function of the MYB84 gene as a negative regulator; its mutation can break the trade-off between yield and drought resistance, achieving synergistic improvement of both traits, providing a completely new technical pathway for rice breeding. Summary of the Invention

[0005] The main objective of this invention is to provide the application of the OsMYB84 gene in regulating rice seed size and drought resistance, for the purpose of regulating rice seed size and drought resistance.

[0006] To achieve the above objectives, the technical solution adopted in this invention is: the application of the OsMYB84 gene in regulating rice seed size and drought resistance. The coding sequence of the OsMYB84 gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2.

[0007] This invention also provides a research method for the application of the OsMYB84 gene in regulating rice seed size and drought resistance. The coding sequence of the OsMYB84 gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2. The method specifically includes the following steps: Step 1: Cloning the rice OsMYB84 gene fragment; Step 2: Overexpress the cloned rice OsMYB84 gene fragment and obtain regenerated plants; Step 3: Construct a knockout vector for the rice OsMYB84 gene and generate mutants; Step 4: Determine the drought resistance of wild plants, regenerated plants, and mutants.

[0008] Preferably, step one specifically includes the following steps: Step 11: Extract total RNA from Zhonghua 11; Step 12: Reverse transcribe total RNA into cDNA; Step 13: PCR amplification of the OsMYB84 gene sequence: A BamHI restriction site was introduced upstream of the OsMYB84 amplification primers, and a SpeI restriction site was introduced downstream. The PCR reaction conditions were: 98℃ for 30 s; 98℃ for 15 s, 60℃ for 15 s, 68℃ for 1 min, for 35 cycles; 68℃ for 10 min. The amplification primers are MYB84-F and MYB84-R. The sequence of MYB84-F is shown in SEQ ID NO:3, and the sequence of MYB84-R is shown in SEQ ID NO:4. Step 14, Electrophoresis detection: After PCR amplification, perform gel electrophoresis to detect whether the OsMYB84 sequence has been amplified; Step 15, DNA recovery from gel cutting: After electrophoresis, OsMYB84 was cut from the gel and the DNA fragments were recovered using a DNA recovery kit.

[0009] Preferably, step two specifically includes the following steps: Step 21: Based on the OsMYB84 gene sequence, pTCK303cGFP was selected as the vector backbone, and BamHI and SpeI were used as restriction enzyme sites to construct the overexpression vector using the enzyme digestion-ligation method. Step 22, Connect the reaction system: The total volume of the reaction system is ,include Carrier skeleton, Purified OsMYB84 gene fragment, T4 DNA ligase, T4 DNA ligase buffer and 2 μL sterile water were added, and the mixture was incubated overnight at 16°C to complete the ligation. Step 23, Conversion and Screening: The ligation products are thermally converted to... The strains were screened for resistance using LB agar plates containing kanamycin; single colonies were picked for colony PCR verification using primers ubi-F and MYB84-R. The recombinant plasmid with correct sequencing was selected and named pTCK303cGFP-OsMYB84 and stored at -20℃ for later use. The sequence of ubi-F is shown in SEQ ID NO:5. Step 24: Take mature seeds of lycopodium (LS), remove the shell and disinfect them, then inoculate them into callus induction medium and culture them in the dark to obtain granular, light yellow embryogenic callus tissue. Step 25: Transform the recombinant vector pTCK303cGFP-OsMYB84 into the Agrobacterium strain, pick positive Agrobacterium single colonies, and culture them in the logarithmic phase; mix rice callus tissue with Agrobacterium bacterial solution and infect for 15-20 min, aspirate excess bacterial solution, inoculate into co-culture medium, and incubate in the dark at 25℃ for 3 days; Step 26: Transfer the co-cultured callus to a selection medium containing hygromycin and discard the untransformed callus; transfer the resistant callus to a differentiation medium and culture under light to induce shoot differentiation; when the shoots grow to 2-3 cm, transfer them to a rooting medium to induce rooting and obtain regenerated plants. Select two regenerated plants with the expected expression levels and growth conditions and name them OsMYB84OE-1 and OsMYB84OE-2 for later use.

[0010] Preferably, step three specifically includes the following steps: Step 31: Using CRISPR design tools, input the OsMYB84 gene sequence, identify and arrange suitable target sites, and calculate and predict the off-target sites for each specified target. Step 32: Select the T1 target site for knockout to obtain the CRISPR-Cas9 plasmid and the knockout vector. The target sequence is as follows: The T1 target sequence is shown in SEQ ID NO:8; Step 33: Obtain mutants Osmyb84-1 and Osmyb84-2 using the obtained knockout vector.

[0011] Preferably, step four specifically includes the following steps: Step 41: Take 150 plump and normal seeds each from wild type, overexpression lines OsMYB84OE-1 and OsMYB84OE-2, and mutants Osmyb84-1 and Osmyb84-2. Step 42: Disinfect the surface of the seeds with 3% sodium hypochlorite for 20 minutes, then rinse the seeds three times with clean water to remove all impurities from the seed surface, and then place them in a 37℃ incubator for germination. Step 43: After the seeds show white sprouts, sow them evenly in the flowerpot. Divide the flowerpot into two halves. Plant the wild type in one half and the overexpression line or mutant in the other half. Divide each type of seed into three replicates. Each replicate contains about 40 seeds. Place them in an artificial climate chamber with a daytime temperature of 30°C, a nighttime temperature of 25°C, and a 16 / 8 light cycle for cultivation. Step 44: When the plants grow to the three-leaf-one-heart stage, start the water-stopping treatment for 10-20 days. If a certain lineage dries out, rehydrate and grow for 7 days, and then count the survival rate. Step 45: Determine the drought resistance of the OsMYB84 gene based on the survival rate.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention has discovered that the OsMYB84 gene can increase the size of rice seeds and enhance the drought resistance of rice. Attached Figure Description

[0013] Figure 1 Image showing the identification of rice OsMYB84 gene overexpression materials; Figure 2 Image showing the seed size results of rice OsMYB84 gene overexpression; Figure 3 A comparison diagram of the rice OsMYB84 gene and two mutant genes; Figure 4 Seed size results for wild type and two mutants; Figure 5 The figure shows the drought resistance results of rice OsMYB84 gene material. Detailed Implementation

[0014] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0015] The coding sequence of the OsMYB84 gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:2.

[0016] I. Cloning the rice OsMYB84 gene, specifically including the following steps: Step 11: Extract total RNA from Zhonghua 11 using TRIZOL; Step 12: Reverse transcription of total RNA into cDNA: Total RNA was reverse transcribed into cDNA using the SuperScript™ III first-strand synthesis system (Invitrogen, America); Step 13: Design primers and perform PCR amplification: A BamHI restriction site was introduced upstream of the OsMYB84 amplification primers, and a SpeI restriction site was introduced downstream. The OsMYB84 gene sequence was amplified by PCR using KOD Neo plus (Toyobo, Japan). The PCR reaction conditions were: 98℃ for 30 s; 98℃ for 15 s, 60℃ for 15 s, 68℃ for 1 min, for 35 cycles; 68℃ for 10 min. The amplification primers are MYB84-F and MYB84-R, respectively. The sequence of MYB84-F is shown in SEQ ID NO:3, and the sequence of MYB84-R is shown in SEQ ID NO:4. Step 14, Electrophoresis detection: After PCR amplification, gel electrophoresis was performed to detect whether the OsMYB84 sequence was amplified. The result showed a clear band around 1149 bp. Step 15, DNA recovery from gel cutting: After electrophoresis, OsMYB84 was cut from the gel and the DNA fragments were recovered using a DNA recovery kit (TransGold, catalog number EG101-02).

[0017] II. Constructing and obtaining the overexpression vector, specifically including the following steps: Step 21: Based on the cloned OsMYB84 gene sequence, pTCK303cGFP was selected as the vector backbone, and BamHI and SpeI were used as restriction enzyme sites to construct the overexpression vector using the enzyme digestion-ligation method. Step 22, Connecting the reaction system: The total volume of the connecting system is ,include Carrier skeleton, Purified OsMYB84 gene fragment, T4 DNA ligase, T4 DNA ligase buffer and 2 μL sterile water were added, and the mixture was incubated overnight at 16°C to complete the ligation. Step 23, Conversion and Screening: The ligation products are thermally converted to... The strains were screened for resistance using LB agar plates containing kanamycin. Single colonies were picked for colony PCR verification using primers ubi-F and MYB84-R. The sequence of ubi-F is shown in SEQ ID NO:5. The PCR program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, and 72℃ extension for 45 s, for a total of 30 cycles; final extension at 72℃ for 5 min; and storage at 4℃. The PCR-positive colonies were sequenced, and the correctly sequenced recombinant plasmid was selected and named pTCK303cGFP-OsMYB84, and stored at -20℃ for later use.

[0018] Step 24: Take mature seeds of "Lansheng (LS)," remove the shells and disinfect them, then inoculate them into callus induction medium and culture them in the dark to obtain granular, light yellow embryogenic callus tissue; Step 25: Transform the recombinant vector pTCK303cGFP-OsMYB84 into Agrobacterium EHA105 or GV3101 strain, pick positive Agrobacterium single colonies, and culture them in the logarithmic phase; mix rice callus tissue with Agrobacterium bacterial solution for 15-20 min, aspirate excess bacterial solution, inoculate into co-culture medium, and incubate in the dark at 25℃ for 3 days; Step 26: Transfer the co-cultured callus to a selection medium containing hygromycin (a selection marker for the vector pTCK303cGFP) to discard untransformed callus; transfer the resistant callus to a differentiation medium and induce shoot differentiation under light; when the shoots reach 2-3 cm in length, transfer them to a rooting medium to induce rooting and obtain regenerated plants (T0 generation transgenic seedlings). Take two types of regenerated plants from the T2 generation with stable expression levels, consistent phenotypes, and normal growth, and perform Western blot analysis to detect the presence of GFP in the plants (…). Figure 1 They were named OsMYB84OE-1 and OsMYB84OE-2 for later use.

[0019] III. Constructing the knockout vector and generating mutants: Using the CRISPR design tool (http: / / tools.genome-engineering.org), the OsMYB84 gene sequence was input to identify and arrange suitable target sites, and the off-target sites for each specified target were calculated and predicted. Finally, the T1 target site was selected for knockout, resulting in a CRISPR-cas9 plasmid, which serves as the knockout vector. "Zhonghua 11" (ZH11) was used as the transformation recipient, and the genetic transformation method was consistent with steps 24-26, yielding regenerated plants (T0 generation transgenic seedlings). Genomic DNA was extracted from the transgenic T1 generation plants, and the MYB84 target sequence was amplified using primers MYB84-idF and MYB84-idR. The MYB84 mutant was identified by sequencing. The sequence of MYB84-idF is shown in SEQ ID NO:6, and the sequence of MYB84-idR is shown in SEQ ID NO:7. Sequencing results showed that the two mutants had insertions of 1 and 2 bases, respectively. Figure 2 The mutants were named Osmyb84-1 and Osmyb84-2, and the target sequence is shown in SEQ ID NO:8.

[0020] IV. Determination of drought resistance in rice seeds of OsMYB84 gene overexpression and knockout lines: Take 150 plump and normal seeds each of wild type, overexpression lines OsMYB84OE-1 and OsMYB84OE-2, and knockout lines Osmyb84-1 and Osmyb84-2. Disinfect the seeds with 3% sodium hypochlorite for 20 minutes, then wash them three times with water to remove all impurities. Place them in a 37℃ incubator for germination. Once the seeds show white sprouts, sow them evenly in flowerpots. To ensure uniform moisture, divide the flowerpots in half. Plant wild type seeds in one half and overexpression or knockout lines in the other half. Each seed type is divided into three replicates, with each replicate containing about 40 seeds. Place them in an artificial climate chamber with a daytime temperature of 30℃, a nighttime temperature of 25℃, and a 16 / 8 photoperiod. When the plants reached the three-leaf-one-heart stage (about 2 weeks), water was withheld for 10-20 days. For one particular line, when it was almost completely dry, it was rehydrated for 7 days. The survival rate was then tallied. The results showed that the survival rate of the overexpression lines was significantly lower than that of the wild type. Figure 5 (A and B), while the survival rate of the mutant lines was significantly higher than that of the wild type ( Figure 5 (C and D) indicates that MYB84 negatively regulates drought resistance in rice.

[0021] V. Determination of seed size in rice OsMYB84 gene overexpression and knockout lines: Plump seeds were collected from wild-type rice plants that were growing normally in the field and harvested, as well as from overexpressing lines of OsMYB84-1 and OsMYB84-2 and knockout lines Osmyb84-1 and Osmyb84-2. At least 12 plants were collected from each line, with at least 100 plump seeds per plant. Grain length, grain width, and thousand-grain weight were then collected using a digital rice seed testing machine (Wuhan Gufeng Optoelectronics, model YTS-5DS). The results showed that the seeds of the MYB84 overexpressing lines were significantly smaller, with significantly reduced grain length, grain width, and thousand-grain weight compared to the wild type. Figure 2 The mutant seeds showed increases in seed length, seed width, and thousand-seed weight compared to the wild type. Figure 4 This indicates that MYB84 negatively regulates rice seed size.

[0022] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. Application of OsMYB84 gene in regulating drought resistance in rice. The coding sequence of OsMYB84 gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:

2.

2. Application of OsMYB84 gene in regulating rice seed size. The coding sequence of OsMYB84 gene is SEQ ID NO:1, and the amino acid sequence is SEQ ID NO:

2.

3. A research method for the application of the OsMYB84 gene in regulating drought resistance in rice, wherein the coding sequence of the OsMYB84 gene is SEQ ID NO:1 and the amino acid sequence is SEQ ID NO:2, and the method specifically includes the following steps: Step 1: Cloning the rice OsMYB84 gene fragment; Step 2: Overexpress the cloned rice OsMYB84 gene fragment and obtain regenerated plants; Step 3: Construct a knockout vector for the rice OsMYB84 gene and generate mutants; Step 4: Determine the drought resistance of wild plants, regenerated plants, and mutants.

4. The research method according to claim 2, characterized in that, Step one specifically includes the following steps: Step 11: Extract total RNA from Zhonghua 11; Step 12: Reverse transcribe total RNA into cDNA; Step 13: PCR amplification of the OsMYB84 gene sequence: A BamHI restriction site was introduced upstream of the OsMYB84 amplification primers, and a SpeI restriction site was introduced downstream. The PCR reaction conditions were: 98℃ for 30 s; 98℃ for 15 s, 60℃ for 15 s, 68℃ for 1 min, for 35 cycles; 68℃ for 10 min. The amplification primers are MYB84-F and MYB84-R. The sequence of MYB84-F is shown in SEQ ID NO:3, and the sequence of MYB84-R is shown in SEQ ID NO:

4. Step 14, Electrophoresis detection: After PCR amplification, perform gel electrophoresis to detect whether the OsMYB84 sequence has been amplified; Step 15, DNA recovery from gel cutting: After electrophoresis, OsMYB84 was cut from the gel and the DNA fragments were recovered using a DNA recovery kit.

5. The research method according to claim 3, characterized in that, Step two specifically includes the following steps: Step 21: Based on the OsMYB84 gene sequence, pTCK303cGFP was selected as the vector backbone, and BamHI and SpeI were used as restriction enzyme sites to construct the overexpression vector using the enzyme digestion-ligation method. Step 22, Connect the reaction system: The total volume of the reaction system is ,include Carrier skeleton, Purified OsMYB84 gene fragment, T4 DNA ligase, T4 DNA ligase buffer and 2 μL sterile water were added, and the mixture was incubated overnight at 16°C to complete the ligation. Step 23, Conversion and Screening: The ligation products are thermally converted to... The strains were screened for resistance using LB agar plates containing kanamycin; single colonies were picked for colony PCR verification using primers ubi-F and MYB84-R. The recombinant plasmid with correct sequencing was selected and named pTCK303cGFP-OsMYB84 and stored at -20℃ for later use. The sequence of ubi-F is shown in SEQ ID NO:

5. Step 24: Take mature seeds of Cymbidium goeringii, remove the shells and disinfect them, then inoculate them into callus induction medium and culture them in the dark to obtain granular, light yellow embryogenic callus tissue. Step 25: Transform the recombinant vector pTCK303cGFP-OsMYB84 into the Agrobacterium strain, pick positive Agrobacterium single colonies, and culture them in the logarithmic phase; mix rice callus tissue with Agrobacterium bacterial solution and infect for 15-20 min, aspirate excess bacterial solution, inoculate into co-culture medium, and incubate in the dark at 25℃ for 3 days; Step 26: Transfer the co-cultured callus to a selection medium containing hygromycin and discard the untransformed callus; transfer the resistant callus to a differentiation medium and culture under light to induce shoot differentiation; when the shoots grow to 2-3 cm, transfer them to a rooting medium to induce rooting and obtain regenerated plants. Select two regenerated plants with the expected expression levels and growth conditions and name them OsMYB84OE-1 and OsMYB84OE-2 for later use.

6. The research method according to claim 4, characterized in that, Step three specifically includes the following steps: Step 31: Using CRISPR design tools, input the OsMYB84 gene sequence, identify and arrange suitable target sites, and calculate and predict the off-target sites for each specified target. Step 32: Select the T1 target site for knockout to obtain the CRISPR-Cas9 plasmid and the knockout vector. The target sequence is as follows: The T1 target sequence is shown in SEQ ID NO:8; Step 33: Obtain mutants Osmyb84-1 and Osmyb84-2 using the obtained knockout vector.

7. The research method according to claim 4, characterized in that, Step four specifically includes the following steps: Step 41: Take 150 plump and normal seeds each from wild type, overexpression lines OsMYB84OE-1 and OsMYB84OE-2, and mutants Osmyb84-1 and Osmyb84-2. Step 42: Disinfect the surface of the seeds with 3% sodium hypochlorite for 20 minutes, then rinse the seeds three times with clean water to remove all impurities from the seed surface, and then place them in a 37℃ incubator for germination. Step 43: After the seeds show white sprouts, sow them evenly in the flowerpot. Divide the flowerpot into two halves. Plant the wild type in one half and the overexpression line or mutant in the other half. Divide each type of seed into three replicates. Each replicate contains about 40 seeds. Place them in an artificial climate chamber with a daytime temperature of 30°C, a nighttime temperature of 25°C, and a 16 / 8 light cycle for cultivation. Step 44: When the plants grow to the three-leaf-one-heart stage, start the water-stopping treatment for 10-20 days. If a certain lineage dries out, rehydrate and grow for 7 days, and then count the survival rate. Step 45: Determine the drought resistance of the OsMYB84 gene based on the survival rate.

Images