Application of OsTAF12 gene in regulating salt tolerance in rice

Abstract

This invention belongs to the field of modern agricultural technology, specifically involving OsTAF12 Application of genes in regulating salt tolerance in rice. This invention discovers... OsTAF12 Genes play a significant role in regulating the salt tolerance of rice. Knocking out rice genes using CRISPR / Cas9 gene editing technology... OsTAF12 And it was knocked out OsTAF12 The homozygous mutant, through a series of experiments, demonstrated that compared to the wild type, OsTAF12 Gene knockout mutants exhibit better salt tolerance phenotypes. Therefore, this invention provides a new method for breeding salt-tolerant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural technology, specifically involving OsTAF12 Application of genes in regulating salt tolerance in rice. Background Technology

[0002] The global area of ​​saline-alkali land is approximately 800 million hectares, and salt stress is one of the major abiotic stresses limiting plant growth and crop yield. Rice ( Rice Rice (Lysimachia christinae) is one of the world's most important food crops, but it is highly sensitive to salt stress. Saline-alkali land resources within global rice-growing areas have enormous development potential.

[0003] Therefore, improving the salt tolerance of rice is of great practical significance for promoting the agricultural utilization of saline-alkali land and increasing rice yield. It is necessary to further study the regulatory genes related to rice salt tolerance and develop new methods for breeding salt-tolerant rice varieties. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention provides suppression OsTAF12 The application of genes in improving salt tolerance in rice, among which OsTAF12 The nucleotide (CDS) sequence of the gene is shown in SEQ ID NO: 2.

[0006] In some embodiments of the present invention, the suppression OsTAF12 Gene methods include knockout OsTAF12 Genes, knockdown OsTAF12 Genes, or make OsTAF12 Methods to induce inactivation mutations.

[0007] A second aspect of the invention provides for suppressing OsTAF12 The application of gene-related biomaterials in rice breeding, among which OsTAF12 The genome sequence of the gene is shown in SEQ ID NO: 1.

[0008] In some embodiments of the present invention, the application is to construct and suppress OsTAF12 Gene-related biological materials were used to obtain rice varieties with improved salt tolerance.

[0009] In some embodiments of the present invention, the biological material does not include reproductive material.

[0010] In some embodiments of the present invention, the biomaterial includes nucleic acid molecules, carriers, and cells.

[0011] In some embodiments of the present invention, the nucleic acid molecule includes an inhibitor. OsTAF12 Functional microRNAs, siRNAs, shRNAs, dsRNAs, sgRNAs, and / or antisense oligonucleotides.

[0012] In some embodiments of the present invention, the sequence of the sgRNA is shown in SEQ ID NO: 4.

[0013] In some embodiments of the present invention, the sgRNA is used in conjunction with a CRISPR / Cas9 vector to achieve gene knockout.

[0014] In some embodiments of the present invention, the CRISPR / Cas9 vector further includes an expression vector containing the sgRNA, and may also include Cas9 protein or an expression vector for expressing Cas9 protein.

[0015] In some embodiments of the present invention, the carrier includes, but is not limited to, other carriers commonly used in the art such as Cas9-MH.

[0016] In some embodiments of the present invention, the cells include at least one of *Escherichia coli* and *Agrobacterium*. *Escherichia coli* is a commonly used host cell in the art for constructing vectors and plasmids, while *Agrobacterium* is a common tool in the art for delivering DNA molecules to plants.

[0017] A third aspect of the present invention provides a method for breeding highly salt-tolerant rice varieties, comprising reducing the salt content of rice. OsTAF12 Steps for determining gene expression levels and / or activity.

[0018] In some embodiments of the present invention, the rice variety includes the following feature: increased salt tolerance relative to a reference level; the reference level is the level of the wild type.

[0019] In some embodiments of the present invention, the reduction of rice OsTAF12 The step of adjusting gene expression levels and / or activity is to combine the second aspect of the invention with the inhibition... OsTAF12 Gene-related biological materials are introduced into rice tissues or rice cells.

[0020] In some embodiments of the present invention, the introduction method includes using at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

[0021] In some embodiments of the present invention, the content of rice is reduced. OsTAF12 The specific steps for determining protein expression levels and / or activity are as follows:

[0022] (1) Design OsTAF12 The target sequence sgRNA of the gene was used to construct rice. OsTAF12Gene-editing CRISPR / Cas9 vector.

[0023] (2) Transform the CRISPR / Cas9 vector described in step (1) into Agrobacterium competent cells to obtain Agrobacterium containing the CRISPR / Cas9 vector.

[0024] (3) Infect the cotyledons of common wild-type rice with the Agrobacterium infection solution obtained in step (3), and re-obtain seedlings through tissue culture to screen rice varieties. OsTAF12 Stable genetic mutant lines that have gene mutations, do not contain exogenous Cas9 protein, and have variations in the target sequence.

[0025] In some embodiments of the present invention, the carrier is Cas9-MH.

[0026] In some embodiments of the present invention, the host cell is Agrobacterium EHA105.

[0027] In some embodiments of the present invention, the rice variety is Zhonghua 11.

[0028] The beneficial effects of this invention are:

[0029] This invention discovers OsTAF12 Genes play a significant role in regulating the salt tolerance of rice. Knocking out rice genes using CRISPR / Cas9 gene editing technology... OsTAF12 And it was knocked out OsTAF12 The homozygous mutant, through a series of experiments, demonstrated that compared to the wild type, OsTAF12 Gene knockout mutants exhibit better salt tolerance phenotypes. Therefore, this invention provides a new method for breeding salt-tolerant rice varieties. Attached Figure Description

[0030] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0031] Figure 1 for OsTAF12 The results of the system evolution analysis.

[0032] Figure 2 for OsTAF12 Gene structure, knockout target information, and mutant identification results.

[0033] Figure 3 for OsTAF12 Phenotypic results of mutants under salt stress, where A represents growth status and B represents the statistical results of survival rate changes over time. Detailed Implementation

[0034] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0035] Example 1: Construction and Identification of the CRISPR / Cas9-OsTAF12 Vector

[0036] This invention is aimed at OsTAF12 Gene validation of its application in rice salt tolerance is publicly available in technical literature in this field. OsTAF12b It may be involved in the mechanisms of abiotic stress in rice, but OsTAF12b (LOC_Os01g62820) and the OsTAF12 discovered in this invention ( LOC_Os01g63940 Although both contain a C-terminal histone-like H2B domain, phylogenetic analysis reveals that they belong to completely different gene branches. Figure 1 ).

[0037] The key differences are as follows:

[0038] 1) Genetic taxonomic evidence: The phylogenetic tree constructed using MEGA 7.0 shows that... OsTAF12 and OsTAF12b They belong to two separate evolutionary branches: OsTAF12b With wheat, corn and other grasses TAF12b They cluster together, exhibiting typical characteristics. TAF12b Subfamily characteristics; OsTAF12 This forms an independent branch, whose evolutionary distance is much greater than that between species. TAF12b Conservatism, its functional differentiation has transcended species differences.

[0039] 2) Differentiation time point: The differentiation of the two occurred earlier than the speciation of monocots and dicots, indicating that... OsTAF12 Not OsTAF12b These are not collateral homologous genes, but independent gene lineages established in the early stages of plant evolution.

[0040] OsTAF12 The genome sequence is as follows:

[0041]

[0042] OsTAF12 The CDS sequence is:

[0043]

[0044] The OsTAF12 protein sequence is as follows:

[0045] N-terminal-MDAPPPAQPDAAAAAAPAAPPTSTSASAPSSAPQPNPTPSASTAAPPTPDTTLAPA PNPTPAPVQTLETPAPSPASARPPVPRMRPPYTHLASPITMSSSPATGAASSSSASAPAATSASSSAMPRGGVALGLPAHPRAPQTPVGYTGFVPPPTLAAQFGSMHRGPDQPPPSSTQPRQP SPGIQNIGTVGSINTSQVRPGAISSLPQQTRPNFPSSTAPSPSDSQIASSQKTPIQALARPPSMASSPSMPLQQTPPNVSAPLRPPQHRPHPRPYHAPAISHPQNALLTQQQQKLPQHQHLQQQ QQQQQQQQKLQQQQQQQQQQKLQQQQQQQQQKLQQQQQQQQNQPQHSSQQSQQTTTLRNQQQISQQQTARTPVSMAQKLDSPAVLKATNVQSGDMASVDVDAGGSGNRLLSKRSIHELVAQIDP SEKLDPEVEDVLIDIAEDFVESVATFACSLAKHRKSSILEAKDVLLHAERSWNITLPGFSGDEIKLYKKPHVNDIHRERLTLIKKSMASESNAKGSAAQAAANQKNQTPKPPATGSP-C terminus (SEQ ID NO: 3).

[0046] To achieve rice OsTAF12 To eliminate gene function, a targeted knockout vector was constructed using CRISPR / Cas9 gene editing technology. OsTAF12 The CDS sequence was used to screen for a 20 bp high-efficiency target in exon 2. The sgRNA sequence is as follows:

[0047] sgRNA: CGCGCCTCCGCCCGCGCAAC (SEQ ID NO: 4).

[0048] The following primers were designed to target the sgRNA-specific primers:

[0049] gRT1: CGCGCCTCCGCCCGCGCAACgttttagagctagaaat (SEQ ID NO: 5);

[0050] OsU3T1:GTTGCGCGGGCGGAGGCGCGTgccacggatcatctgc (SEQ ID NO: 6).

[0051] BL:

[0052] ACCGGTAAGGCGCGCCGTAGTGCTCGACTAGTATGGAATCGGGCAGCAAAGG (SEQ ID NO: 7);

[0053] BR:

[0054] TAGCTCCGAGAGGCGCCAATGATACCGACGCGTATCCATCCACTCCAAGTCTTG (SEQ ID NO: 8).

[0055] Using OsU3 plasmid diluted 20-fold as a template, PCR amplification was performed using BL / gRT1 and OsU3T1 / BR primers, respectively. The products were mixed in equal proportions and diluted 10-fold as templates for a second round of PCR amplification using BL / BR primers to obtain fragments containing complete gRNA structures.

[0056] Subsequently, the PCR product was inserted into the CRISPR / Cas9 backbone vector Cas9-MH using a Golden Gate ligation reaction. The ligation reaction system is shown in Table 1.

[0057] Table 1

[0058]

[0059] The reaction conditions were: 37℃ for 5 min, 16℃ for 5 min, for 15 cycles. After the reaction, 15 μL of the product was transformed into competent Escherichia coli DH5α, and LB plates containing 50 μg / mL kanamycin were selected. Positive clones were then identified by colony PCR and sequencing. Those correctly sequenced were named the CRISPR / Cas9-OsTAF12 expression vector.

[0060] The construction process referenced relevant literature (Ma XL, et al. 2015). A robust CRISPR / Cas9 system for convenient high-efficiency multiplex genome editing in monocot and dicotplants. Molecular Plant , 8(8):1274–1284.).

[0061] Example 2: CRISPR / Cas9-OsTAF12 transformation of rice and screening of positive lines

[0062] 1. Agrobacterium-mediated transformation

[0063] The successfully constructed Cas9-OsTAF12 vector was transformed into Agrobacterium tumefaciens EHA105 competent cells. Positive strains were identified by PCR and then used for further processing. Single colonies were inoculated into liquid medium containing kanamycin (100 μg / mL) and rifampin (50 μg / mL) and cultured at 28°C with shaking. Cells reaching an OD600 ≈ 0.8–1.0 were then used to infect callus tissue of Zhonghua 11.

[0064] Transformation was performed using Agrobacterium-mediated transformation, which included infection, co-culture, resistance selection, shoot differentiation, and rooting culture to obtain T0 generation transgenic plants. The rice transformation method was referenced in the relevant literature (Zhao W, Zheng S, Ling HQ (2011). An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297. Plant Cell TissOrgan Cult, 106:475–483.).

[0065] 2. Identification of T0 generation plants of OsTAF12 mutant

[0066] Genomic DNA was extracted from T0 generation plants, and the following primers were used for PCR amplification and sequencing analysis of the target sites:

[0067] OsTAF12gF: TAGGCAGATGAAGGCAACTC (SEQ ID NO: 9);

[0068] OsTAF12gR: AGCTGGGGTTTCTAGGGTTT (SEQ ID NO: 10).

[0069] After sequencing the PCR amplification products, the sequences were compared with those of the wild-type (ZH11) line to identify two homozygous mutant lines:

[0070] Ostaf12-51 The insertion of a "T" base at the target site leads to a frameshift mutation.

[0071] Ostaf12-53 The absence of the two "GC" bases at the target site leads to a frameshift mutation.

[0072] Example 3 Salt tolerance analysis of OsTAF12 gene knockout lines

[0073] Under greenhouse hydroponic conditions, the wild-type Zhonghua 11 (ZH11) and OsTAF12 knockout lines ( Ostaf12-51 and Ostaf12-53 Salt tolerance was verified. The experimental procedure is as follows:

[0074] 1. Hydroponic seedling cultivation and salt stress treatment

[0075] The seeds were sterilized with 10% H2O2 for 10 min, and after removing the residue, they were placed on sterile filter paper and pre-cultured at 37℃ in the dark for 2 days, followed by germination at 28℃ for 1 day.

[0076] Germinated seeds were transferred to a hydroponic box and cultured for 7 days, with the nutrient solution changed every 2 days.

[0077] The growth chamber conditions were: 28℃ / 25℃ (day and night), relative humidity 70%, photoperiod 14 h / 10 h, and light intensity 400 μmol·m⁻²·s⁻¹;

[0078] When the plant grows to the 3-leaf stage, add 120 mM NaCl to implement salt stress treatment for 6 days.

[0079] The nutrient solution formula is shown in Table 2.

[0080] Table 2

[0081]

[0082] 2. Phenotypic observation and data analysis

[0083] Six days after salt stress treatment, wild-type plants showed obvious symptoms such as wilting, curling, and water loss, while the two knockout lines... Ostaf12-51 and Ostaf12-53 It exhibited stronger growth vigor and stress resistance, with a significantly higher survival rate than ZH11, increasing by 33.4% and 36.7%, respectively.

[0084] The results showed that knocking out the OsTAF12 gene significantly enhanced the rice's tolerance to salt stress.

[0085] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Inhibition OsTAF12 The application of gene-based reagents in breeding highly salt-tolerant rice varieties is characterized by: The OsTAF12 The nucleotide sequence of the gene is shown in SEQ ID NO: 2; The reagent is 1) Nucleic acid molecules; The nucleic acid molecules include knockout OsTAF12 CRISPR / Cas9 vectors and sgRNAs for gene function; The sequence of the sgRNA is shown in SEQ ID NO: 4; or 2) Cells containing the nucleic acid molecules described in 1); The cells include Escherichia coli and Agrobacterium.

2. A method for breeding highly salt-tolerant rice varieties, comprising reducing the salt content of rice... OsTAF12 Steps for measuring gene expression levels; in, Rice varieties possess the following characteristics: increased salt tolerance relative to a reference level, where the reference level is the level of the wild type; The reduction of rice OsTAF12 The step of regulating gene expression is to reduce the inhibition described in claim 1. OsTAF12 Gene-generating reagents are introduced into rice tissues or rice cells.

3. The method according to claim 2, characterized in that: The importation method includes at least one of Ti plasmid, Ri plasmid, plant virus vector, microinjection, and electroporation.

Images