Application of OsMYB36 protein and its encoding gene in regulating rice salt tolerance

Abstract

This invention relates to the field of rice genetic engineering, disclosing the application of the OsMYB36 protein and its encoding gene in regulating salt tolerance in rice. This invention uses CRISPR / Cas9 technology to edit the OsMYB36 protein gene, introduces it into the japonica rice variety Zhonghua 11 using Agrobacterium-mediated transformation, and screens for knockout mutants; experimental results demonstrate the effectiveness of the knockout mutant. OsMYB36 The rice mutant of the gene, after being treated with 150 mM NaCl and 200 mM NaCl for 11 days at the 2-leaf-1-heart stage and then rehydrated for 7 days, showed a significantly higher survival rate than the wild-type Zhonghua 11 plants. This provides a simple and effective technical means for rapidly creating new salt-tolerant rice lines. Therefore, the OsMYB36 protein and its encoding gene of this invention can regulate the salt tolerance of rice, which is of great significance for breeding salt-tolerant transgenic rice.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically the application of the OsMYB36 protein and its encoding gene in regulating salt tolerance in rice. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is one of the world's most important food crops. However, throughout its growth process, rice is frequently subjected to various abiotic stresses, such as salinity, drought, flooding, and extreme temperatures. Among these, salinity stress is one of the most significant abiotic stresses threatening rice production.

[0003] Soil salinization has led to a reduction in arable land, contributing to the food crisis. Faced with a growing population, limited available arable land, and increasingly severe secondary soil salinization due to inefficient irrigation, making it difficult to significantly increase rice yields, developing and utilizing coastal and inland saline-alkali land resources is an effective way to ensure arable land availability. Rice is a moderately salt-sensitive crop, growing in aquatic environments; rice cultivation can leach soluble salts and alkalis from the soil. Therefore, rice is the preferred food crop for developing coastal and saline-alkali land.

[0004] Improving salt tolerance in rice through genetic modification is one of the effective ways to increase rice planting area and yield. Currently, the salt tolerance QTLs used in breeding are mainly located on chromosome 1 of rice. qSKC-1 and Saltol Two loci. With the development of molecular biotechnology, using mutants to isolate and discover salt stress-tolerant genes in rice, and applying them to rice genetic engineering for assisted breeding and alkali stress improvement, is of paramount importance for effectively controlling the damage of salt stress to rice, increasing rice yield, and improving rice quality. 59 MYB transcription factors have been cloned in rice. OsMYB41 Positive regulation of rice salt tolerance; OsMYB2 Overexpression of R2R3 MYB transcription factors involved in stress responses OsMYB2 or OsANT1 This results in an increase in the number of tillers per plant, the number of grains per plant, and the yield per plant, as well as improved nitrogen utilization and enhanced salt tolerance. OsMYB6 It is a positive regulator of drought and salt stress responses, and overexpression OsMYB6 It can enhance the drought and salt tolerance of genetically modified rice; OsBAG4 , OsMYB106 and OsSUVH The transcriptional complex composed of these seven genes can regulate the major salt tolerance gene. OsHKT1;5 Expression; mutant osmyb106 It exhibits a salt-sensitive phenotype and displays dwarfing and pollen abortion; OsMYBc Able toOsHKT1;1 The AAANATNC(C / T) sequence of the promoter binds and upregulates OsHKT1;1 This expression, in turn, positively regulates the salt tolerance of rice; OsMYB21 It is an R2R3 type MYB transcription factor that plays a crucial role in seed germination, seedling growth, and root elongation. OsMYB21 Overexpression of genes can increase the salt tolerance of transgenic lines. OsMYB36a , OsMYB36b and OsMYB36c It synergistically regulates lignin deposition in the rice root endodermis, the formation of the Casparian strip, and plays an important role in the selective absorption of nutrients in the roots, but no further research has been found on... OsMYB36 Reports on rice salt stress resistance. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the OsMYB36 protein and its encoding gene in regulating rice salt tolerance and in creating salt-tolerant rice lines.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The first aspect of this invention provides the application of genes that knock out the OsMYB36 protein, said application being any of the following:

[0008] A1) Application in improving salt tolerance in rice;

[0009] A2) Application in the preparation of products that improve the salt tolerance of rice;

[0010] A3) Application in the cultivation of salt-tolerant rice;

[0011] A4) Application in the preparation of products for cultivating salt-tolerant rice;

[0012] A5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources;

[0013] The amino acid sequence of the OsMYB36 protein is shown in SEQ ID NO. 3.

[0014] A second aspect of the invention provides the application of biomaterials related to the OsMYB36 protein, wherein the application is any of the following:

[0015] B1) Application in improving salt tolerance in rice;

[0016] B2) Application in the preparation of products that improve the salt tolerance of rice;

[0017] B3) Application in the cultivation of salt-tolerant rice;

[0018] B4) Application in the preparation of products for cultivating salt-tolerant rice;

[0019] B5) Application in salt-tolerant breeding of rice or improvement of salt-tolerant rice germplasm resources;

[0020] The biomaterial is any one of the following C1) to C3):

[0021] C1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the OsMYB36 protein;

[0022] C2) An expression cassette containing the nucleic acid molecule described in C1);

[0023] C3) A recombinant vector containing the nucleic acid molecule described in C1), or a recombinant vector containing the expression cassette described in C2);

[0024] C4) A recombinant microorganism containing the nucleic acid molecule described in C1), or a recombinant microorganism containing the expression cassette described in C2), or a recombinant microorganism containing the recombinant vector described in C3), wherein the microorganism is Agrobacterium.

[0025] Furthermore, the nucleotide sequence of the nucleic acid molecule encoding the OsMYB36 protein is shown in SEQ ID NO. 2.

[0026] A third aspect of the present invention provides a method for improving the salt tolerance of rice, the method comprising knocking out the gene for the OsMYB36 protein in rice to obtain rice with improved salt tolerance, wherein the amino acid sequence of the OsMYB36 protein is shown in SEQ ID NO.3.

[0027] In the above method, knocking out the OsMYB36 protein gene in rice involves using gene knockout technology to reduce the expression level of the gene encoding the OsMYB36 protein.

[0028] In the above method, the expression level of the gene encoding OsMYB36 protein is reduced by using gene knockout technology to perform targeted knockout of the OsMYB36 protein gene using a CRISPR / Cas9 system containing the target site shown in SEQ ID NO.5.

[0029] The fourth aspect of the present invention provides a method for breeding salt-tolerant rice varieties, comprising: using the salt-tolerant rice obtained by the above method as a parent, hybridizing it with a target material, and backcrossing the obtained F1 generation with the target material so that the backcross offspring acquire the same salt tolerance trait as the parent.

[0030] Furthermore, the salt-tolerant backcross offspring and the parents possess the same mutated gene; OsMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the mutated gene is... OsMYB36The gene has a 13-base deletion from position 92 to position 104 of the start codon ATG, and its nucleotide sequence is shown in SEQ ID NO. 4.

[0031] The beneficial effects of this invention are:

[0032] (1) This invention is the first to discover and confirm that OsMYB36 This gene negatively regulates salt tolerance in rice. Inhibiting or knocking out this gene actually enhances the salt tolerance of rice. This invention provides a new perspective and target for the molecular mechanism of rice's response to salt stress, enriching the theoretical basis of plant salt tolerance.

[0033] (2) Specifically, this invention provides a method for precise knockout using CRISPR / Cas9 gene editing technology. OsSTA230 The gene-based approach includes specific target sites (SEQ ID NO.5) and corresponding targeting vectors. This method is targeted, efficient, and quick, overcoming the bottlenecks of traditional breeding where selecting for salt tolerance traits is difficult and time-consuming.

[0034] (3) Through the above method, we successfully obtained OsMYB36 Gene knockout mutants. Rigorous physiological experiments demonstrated that after treatment with 150 mM and 200 mM NaCl salt stress, the survival rate of the mutants (58.33%) was significantly higher than that of the wild type (8.33%). This provides genetic material and gene resources with direct application value for salt-tolerant rice breeding.

[0035] (4) This invention not only provides a method for creating core salt-tolerant germplasm, but also clarifies a breeding strategy for rapidly introducing this salt-tolerant trait into other superior rice varieties through hybridization and backcrossing. This means that the technological achievement can be quickly applied to actual production to cultivate new rice varieties adapted to saline-alkali land. Attached Figure Description

[0036] Figure 1 for OsMYB36 Schematic diagram of gene structure and target sequence elements of CRISPR / Cas-OsMYB36 vector.

[0037] Figure 2 These are peak diagrams of sequencing results; the top diagram shows the sequencing peaks of the wild-type ZH11 target, and the bottom diagram shows the peaks of the mutant. osmyb36 Target sequencing peak diagram.

[0038] Figure 3 for osmyb36 Salt tolerance test of mutants. A represents ZH11 and... ossgr1 The plant's growth state before salt treatment, bar = 1 cm; B represents ZH11 and osmyb36The growth status of plants after salt treatment, bar = 1 cm; C represents the survival rate statistics of ZH11 and ossgr1 before and after salt treatment. The values ​​shown are mean ± standard deviation, n = 3. * indicates significant difference, P < 0.05; ** indicates extremely significant difference, P < 0.01. The statistical analysis method is one-way ANOVA.

[0039] Figure 4 This is a schematic diagram of the backcrossing and breeding route. Detailed Implementation

[0040] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0041] Unless otherwise specified, the experimental methods described below can be performed using conventional methods in this field. Unless otherwise specified, commercially available materials can be used for the experiments described below.

[0042] CRISPR / Cas vector BGK03: Hangzhou Baige Biotechnology Co., Ltd., product catalog number BGK03. Example 1

[0043] This embodiment provides rice. OsMYB36 The functions and applications of genes include the following:

[0044] 1. Rice OsMYB36 Gene sequence and expression pattern analysis

[0045] The Ensembl Plants database (http: / / plants.ensembl.org / index.html) contained information on rice. OsMYB36 The nucleotide sequence of the gene in *Zhonghua 11* of japonica rice is shown in SEQ ID NO. 1, and the CDS sequence is shown in SEQ ID NO. 2. Its encoded protein contains 563 amino acids, and its sequence is shown in SEQ ID NO. 3. To study the function of this gene, this invention first used a gene expression database to analyze the expression pattern of this gene in different tissues of rice. The expression analysis results showed... OsMYB36 The gene is predominantly expressed in the rice roots, suggesting that it may be involved in the rice root development process.

[0046] 2. Rice OsMYB36 Functional verification of genes

[0047] To clarify OsMYB36To investigate the function of a gene in rice, this invention employs the CRISPR / Cas9 gene editing method to site-directedly mutate the gene sequence and knock out its function in rice. This invention selects the conventional rice variety ZH11 as the recipient material for gene editing. This invention selects 20 bases from position 91 to position 110 of the gene coding region, as shown in SEQ ID NO. 5, as the target region for CRISPR / Cas9 gene editing (see [link to CRISPR / Cas9 gene editing instructions]). Figure 1 ).

[0048] (1) OsMYB36 Construction of CRISPR / Cas9 gene editing vector

[0049] The gene editing vector of this invention is pEGCas9Pubi-B-OsMYB36, and the base vector of this vector is pEGCas9Pubi-B. This invention involves designing target sites on primers, obtaining MT-sgRNA via PCR, and then ligating it into the base vector using a one-step cloning method. The specific construction process is as follows:

[0050] i) Design of target gRNA. [The following is likely a separate, unrelated sentence:] OsMYB36 The gene sequence was input into https: / / zlab.bio / guide-design-resources for target design, and the PAM sequence was set to NGG. The DNA sequence of the target region selected in this invention is shown in SEQ ID NO.5.

[0051] ii) Amplification of the sgRNA expression cassette by overlap PCR and nested PCR. Primer pairs containing the above-mentioned sgRNA target sequences were synthesized and annealed. Then, the primer pairs were ligated with the binary vector pEGCas9Pubi-B (see Ma X, Zhang Q, Zhu Q. et al. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants, Mol Plant. 2015, 8(8):1274-1284, vector pEGCas9Pubi-B was kindly provided by Professor Long Tuan of Hainan University) to obtain the recombinant vector pEGCas9Pubi-OsMYB36. The recombinant vector pEGCas9Pubi-OsMYB36 was transformed into E. coli DH5α, and positive clones were selected for sequencing. The specific steps were as described in the reference "Xing, H.L., Dong, L., Wang, Z.P., Zhang, H.Y., Han, C.Y., Liu, B., Wang, X.C., and Chen, Q.J. (2014). A CRISPR / Cas9toolkit for multiplex genome editing in plants. BMC plant biology 14:327."

[0052] iii) Sequencing verification.

[0053] The successful construction of pEGCas9Pubi-OsMYB36 was verified by sequencing.

[0054] (2) Agrobacterium-mediated genetic transformation of rice

[0055] The constructed pEGCas9Pubi-OsMYB36 vector was transformed into Agrobacterium EHA105 via heat shock. After PCR identification, the bacterial culture was stored at -80 °C with glycerol. Freshly peeled embryos (approximately 1.5 mm in diameter) of the rice hybrid Zhonghua 11 were used as recipient material. The peeled embryos were placed in 2 mL plastic centrifuge tubes containing 1.8 mL of suspension for no more than 1 hour, with approximately 100 embryos per tube. The suspension was removed, and the embryos were washed twice with fresh suspension, leaving a small amount of suspension at the bottom of the tube to submerge the embryos. The tubes were then heat-shocked at 43 °C for 2 minutes, followed by an ice bath for 1 minute. The remaining wash solution at the bottom of the tube was aspirated with a pipette, and 1.0 mL of Agrobacterium infection solution was added. The tubes were gently shaken for 30 seconds and then incubated in the dark for 8 minutes. Next, pour the embryos and infection solution from the centrifuge tubes onto the co-culture medium, shake well, and then use a pipette to remove excess infection solution. Ensure all embryos have their scutellaria facing upwards and co-culture at 23°C in the dark for 3 days. After co-culture, use sterile forceps to transfer the embryos to recovery medium and culture at 28°C for 7-14 days. During this process, carefully remove any sprouts that appear on the embryos. After recovery culture, place the embryos on 1.5 mg / L Bialaphos selection medium for 3 rounds of selection, each round lasting 2 weeks. Then transfer them to 2 mg / L Bialaphos selection medium for 2 rounds of selection, each round lasting 2 weeks. Transfer the resistant callus to propagation medium and culture at 28°C in the dark for 2 weeks. Then transfer the propagated resistant callus to induction medium and culture at 28°C in the dark for 2 weeks. Finally, transfer it to differentiation medium and culture at 25°C, 5000 lx under light for 2 weeks. After the culture is completed, the differentiated seedlings are separated into individual seedlings and placed in a rooting medium. They are cultured at 25 ℃, 5000 lx, and under light until they root. The seedlings are then transferred to small nutrient pots for growth. After they have survived, they are transplanted into a greenhouse. The offspring seeds are harvested 3-4 months later.

[0056] (3) Detection of CRISPR / Cas9 mutation results in T0 generation plants

[0057] To determine the CRISPR / Cas9 mutation results in T0 generation plants, the following steps were taken for detection:

[0058] This invention first employs the CTAB method to extract DNA from rice leaves. The specific method is as follows: DNA extraction is performed according to the traditional CTAB method (Rogers and Bendich, 1985). A 3 cm rice leaf is placed in a sterilized 2 mL centrifuge tube, a 6 mm steel ball is added, and the tissue is disrupted using a cell disruptor. Then, CTAB extraction is performed. Finally, 200 μL of sterile water (ddH2O) is added to dissolve the air-dried DNA sample, which is then set aside. After the DNA is completely dissolved, 2 μL of the sample is taken and the nucleic acid OD value (A260 / A280) and nucleic acid concentration are determined using a UV spectrophotometer (Nanodrop 2000). The DNA sample is then diluted to 50 ng / μL for later use.

[0059] PCR was performed using 5 μL of Biomiga 2×PCR premix (containing Mg2+, Taq DNA Polymerase, 2.5 mM dNTPs, and 10×PCR Buffer), 1 μL of primers (containing 0.5 μL each of forward and reverse primers), 1 μL of template DNA, and ddH2O to a final volume of 10 μL. The PCR amplification program was a standard SSR program (94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles, and a final extension at 72℃ for 5 min). The amplified products were analyzed by 8% non-denaturing polyacrylamide gel electrophoresis, stained with 0.1% AgNO3, and photographed after color development with formaldehyde and NaOH.

[0060] For mutants osmyb36 Nucleotide sequence alignment analysis revealed that ( Figure 1 Compared to the unedited wild type (WT), the mutated OsMYB36 The mutant deletes 13 bases from position 92 to position 104 of the start codon ATG, as shown in SEQ ID NO.4. This nucleotide deletion causes a frameshift in the amino acid sequence, leading to premature termination of translation. Example 2

[0061] This embodiment describes the mutant obtained in Example 1. osmyb36 Phenotypic analysis was performed, as follows:

[0062] 1. Mutant osmyb36 Salt tolerance assessment

[0063] The parameters for alternating light and dark culture are as follows: light intensity is 120 μmol·m⁻¹. -2 ·s -1 The temperature is 28℃ / 25℃ (day / dark), and the photoperiod is 10h light / 14h darkness.

[0064] The rice seeds to be tested are osmyb36 - L1 Homozygous seeds of mutant T1 generation, along with its background material ZH11 and empty vector control. The experiment was repeated three times, and the average value was taken. The steps for each repetition are as follows:

[0065] 1. For each material, take 12 rice seeds to be tested, put them into kraft paper bags, and soak them in water at 28℃~30℃ for 48 hours.

[0066] 2. After completing step 1, germinate the seeds at 28℃~30℃ for 24 hours (keep the seeds moist during germination) to obtain germinated seeds.

[0067] 3. After completing step 2, take a 96-well plate, cut off part of the lower edge of each well, and then put one germinated seed into each well (embryo facing up, radicle facing down).

[0068] 4. After completing step 3, place the 96-well plate (containing the germinated seeds) on a plastic box containing Yoshida rice culture medium, immersing the germinated seeds in the medium. Culture in alternating light and dark conditions for 3 weeks to obtain rice seedlings that have reached the three-leaf stage. During the alternating light and dark culture period, the Yoshida rice culture medium should be replaced every 7 days.

[0069] 5. After completing step 4, place the 96-well plate (containing rice seedlings that have grown to the three-leaf stage) on a plastic box containing 150 mM NaCl Yoshida rice culture solution and ensure that the roots are completely immersed in the culture solution. After 3 days of treatment, replace the Yoshida rice culture solution with 200 mM NaCl and treat again. After 3 days, replace the Yoshida rice culture solution with 150 mM NaCl and treat again. Perform high salt stress for 11 days under alternating light and dark conditions (during the high salt stress period, replace the Yoshida rice culture solution every 2 days).

[0070] 6. After completing step 5, place the 96-well plate (with rice seedlings on it) on a plastic box containing Yoshida rice culture medium and allow it to recover for 7 days under alternating light and dark conditions.

[0071] Observe the growth status of rice seedlings and calculate the survival rate. Survival rate = (Number of surviving rice seedlings / 12) × 100%.

[0072] See the growth status of rice seedlings before treatment. Figure 3 The growth status after treatment A is shown in the figure. Figure 3 Survival rate statistics for B are shown in [link to data]. Figure 3 C.

[0073] The results showed that before salt treatment, osmyb36 The growth pattern was basically the same as that of ZH11; freshwater control ZH11 and mutantosmyb36 The survival rate was 100%. After salt solution treatment, the survival rate of ZH11 was 8.33%, while that of the mutant... OsMYB36 The survival rate was 58.33%, and statistical analysis results showed that... osmyb36 The survival rate of [the strain] was significantly higher than that of ZH11, indicating that... osmyb36 The salt tolerance of the mutant was significantly improved. The phenotype and survival rate of the empty vector control were basically consistent with those of the background material ZH11, with no statistical difference. Example 3

[0074] This embodiment performs osmyb36 Transgenic experiments using mutant genes, using mutants osmyb36 By crossing, backcrossing, and self-pollinating R3261 with a recipient variety exhibiting excellent agronomic traits, restorer lines carrying homozygous mutant genes in the R3261 background were ultimately obtained. The technical route for hybridization and conversion is as follows: Figure 4 As shown.

[0075] The above example uses R3261 as a breeding example, but it is not limited to R3261 and can be any rice material.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of gene knockout of OsMYB36 protein in improving salt tolerance in rice, wherein the amino acid sequence of the OsMYB36 protein is shown in SEQ ID NO.3; The mutated gene... OsMYB36 The nucleotide sequence of the gene is shown in SEQ ID NO.

4.

2. A method for improving the salt tolerance of rice, characterized in that, The method involves knocking out the gene for the OsMYB36 protein in rice to obtain a mutant with the nucleotide sequence shown in SEQ ID NO.

4. OsMYB36 The gene, thereby obtaining rice with improved salt tolerance; the amino acid sequence of the OsMYB36 protein is shown in SEQ ID NO.

3.

3. The method according to claim 2, characterized in that, The gene that knocks out the OsMYB36 protein in rice is obtained by using gene knockout technology to reduce the expression level of the gene encoding the OsMYB36 protein.

4. The method according to claim 3, characterized in that, The reduction in the expression level of the gene encoding OsMYB36 protein using gene knockout technology is achieved by targeted knockout of the OsSTA230 protein gene using the CRISPR / Cas9 system, the target site of which is shown in SEQ ID NO.5.

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