MYB transcription factor related to rice salt tolerance

Abstract

The invention belongs to the technical field of agricultural biology, and relates to application of a transcription factor MYB41 with an amino acid sequence of SEQ ID NO: 1 to improvement of plant salt tolerance, which is beneficial to plant repair, salt-tolerant plant germplasm resource creation and salt-tolerant rice variety cultivation.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to MYB41, a MYB family transcription factor related to salt tolerance in rice, and its application in improving plant salt tolerance and creating salt-tolerant plants. Background Technology

[0002] Soil salinization is one of the major agricultural problems facing the world. Since rice is an important food crop, understanding the mechanisms by which rice responds to salt stress is particularly important. The effects of salt stress on plants are mainly manifested in three aspects: first, by inducing osmotic stress, limiting plant water absorption; second, by disrupting ion balance through the absorption of toxic sodium and chloride ions; and third, by inhibiting plant growth and promoting senescence by disrupting redox homeostasis. Therefore, rice's regulation of salt stress mainly involves osmotic regulation, ion homeostasis, antioxidant system regulation, and nutrient regulation.

[0003] As sessile and autotrophic organisms, plants cannot escape the fluctuations in nutrient availability and toxicity in their environment. Plant roots are responsible for nutrient acquisition, but they must also provide an effective boundary to resist external stresses and maintain plant health. The endodermis of plant roots has two main specialized structures: lignified banded structures and suberized lamellae surrounding the entire endodermal cell. These two structures enclose the endodermis outside the stele, forming a barrier for root water and mineral elements, protecting the vascular system used for transport in the plant roots. The lignified banded structures develop earlier, while the suberized structures develop later. These endodermal barriers are considered screens in the pathway from the soil to the plant's vascular tissue. Numerous articles have reported that suberization occurs prematurely in plant roots after salt treatment. Suberization refers to the deposition of suberin in the cell wall, which encapsulates the entire cell. Suberin acts as an apoplast "barrier" in the endoderm and epidermis of plant roots, playing a role in ultrafiltration of sodium ions. As a "barrier," suberin has been shown to potentially participate in the salt tolerance function of plants.

[0004] In the regulatory mechanisms of suberin, a series of transcription factors of the MYB family play a key role. MYB transcription factors are a group of pan-eukaryotic transcription factors, defined as those containing one to four MYB repeat sequences; each MYB repeat sequence consists of 50 amino acids in three α-helices. Based on the number of MYB repeats and the identity of the MYB sequence, MYB proteins are usually classified into MYB-related proteins, R2R3-MYB, R1R2R4-MYB, and 4R-MYB proteins, which are crucial for their DNA binding [JiangCK, RaoGY, 2020]. First, researchers identified a MYB family transcription factor, MYB41. Overexpression of this gene can activate aliphatic suberin synthesis and cell wall-associated suberin lamellar deposition in Arabidopsis and tobacco. Gas chromatography-mass spectrometry (GC-MS) detected increased suberin monomer content in both Arabidopsis and tobacco plants overexpressing AtMYB41, leading to a 22-fold increase in the accumulation of fatty suberin monomers in leaves compared to cutin aliphatic monomers, such as long-chain fatty acids, ω-hydroxy acids, α-diacids, ω-diacids, and fatty alcohols. Transmission electron microscopy also revealed a distinct suberin lamellar structure [Kosma DK, 2014]. Further analysis showed that AtMYB41 overexpression increased the abundance of suberin biosynthetic gene transcripts (such as FAR, GPAT, ASFT, CYP86, and PER) by an order of magnitude, providing a deeper understanding of the ubiquitous suberin structure and the molecular genetic mechanisms of its biosynthesis. Recently, a series of MYB family transcription factors have been identified. Firstly, in the identification of AtMYB41, analysis of single-gene mutant lines of MYB41 revealed no significant reduction in suberin monomers, disappearance of suberin lamellae, or downregulation of genes related to suberin synthesis. This suggests a severe redundancy of genes in the MYB family regarding suberin synthesis. However, in the myb41myb53myb92myb93 quadruple mutant, suberin in Arabidopsis roots cannot be deposited, even under ABA or CIF peptide induction or salt stress [Shukla V.2021]. Mass spectrometry revealed a significant decrease in suberin fatty acid monomers compared to the wild type, and downregulation of genes related to suberin biosynthesis. Ionomic phenotype also confirmed the absence of the endodermal barrier. Other researchers have used estradiol-induced systems to demonstrate that overexpression of a series of MYB transcription factors, MYB6, MYB9, MYB39, MYB68, MYB74, MYB84, MYB92, MYB93, and MYB122, can affect the initiation position of the Casparian strip and the suberization of the endothelial layer to varying degrees.In addition, researchers used yeast one-hybrid technology and qRT-PCR technology to establish a gene regulatory network mediated by SHR transcription factors, which ultimately affects the endothelial barrier function and balances endothelial lignification and corkation.

[0005] To enable plants to adapt to soil salinization, to find rice varieties that can be grown in saline-alkali land, and to elucidate the molecular mechanism of salt tolerance in rice suberin, are of great theoretical and practical significance for the breeding of new rice varieties. Summary of the Invention

[0006] In our research on rice salt stress tolerance technology, we investigated the role of MYB family transcription factors in regulating the biosynthesis of suberin molecules. We found that one transcription factor, MYB41 (NCBI ID Os04g0593200), plays a positive role in maintaining normal plant growth and improving rice salt stress tolerance. Loss-of-function mutants of MYB41 exhibited poor growth phenotypes and were sensitive to salt, indicating that Os04g0593200 positively regulates rice growth and salt tolerance. Furthermore, we discovered a promoter that promotes the expression of the Os04g0593200 gene. Based on these research findings, this invention includes the following technical solution.

[0007] The first aspect of this invention provides the application of transcription factor MYB41 (NCBI number Os04g0593200) or its expression gene MYB41, with the amino acid sequence shown in SEQ ID NO:1, in improving plant salt tolerance:

[0008] MGRSPCCCHDAGVKKGPWTEEEDRALVEHIKKQGGHVGSWRGLPRAAGLNRCGKSCRLRWTNYLRPDIRRGNFSDDEERLIIRLHAALGNKWSTIATHLDGRTDNEIKNYWNTHIKKKLLRMGIDPVTHQRLPPDLLADGGGLGAASPLLSPPGPAAAAALQPLLSAVASLG SLDTALRQFQLLQHLLNSITSSSSDVAATAGLMATNLAATNTMVNSSSSNVASFQEQMNALAHANYQPGYLRDVVPSFPGQDMAPQLNSTSSTPSTAPVLRSSAEPADQCCNDAALVPETYPREVAASVDHWKVQDFPSLEPLELPNLSTLESDLDPFWKEILESSFRS(SEQ ID NO:1).

[0009] The plants mentioned above are preferably monocotyledonous plants, such as grass crops, and can be selected from rice, wheat, corn, soybean, barley, oats, rye and sorghum.

[0010] Preferably, the crop mentioned above is rice.

[0011] In one embodiment, the nucleotide sequence of the expression gene MYB41 of the above-mentioned transcription factor MYB41 (NCBI number Os04g0593200) is SEQ ID NO:2.

[0012] In the above-mentioned application methods, the creation of salt-tolerant plant germplasm or the breeding of salt-tolerant plant varieties are carried out by overexpressing the transcription factor MYB41 (Os04g0593200) or its expression gene MYB41 in plants.

[0013] In the above application method, the overexpression of transcription factor MYB41 (Os04g0593200) or its expression gene MYB41 is achieved in the following manner:

[0014] A. The gene MYB41 with the nucleotide sequence SEQ ID NO:2 was cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely the MYB41 overexpression vector. Plants were then transformed using Agrobacterium-mediated transformation to obtain transgenic plants overexpressing the transcription factor MYB41 (Os04g0593200); or

[0015] B. By cloning the gene MYB41 with the nucleotide sequence SEQ ID NO:2 into a plant chromosome using gene editing technology, transgenic plants overexpressing the transcription factor MYB41 (Os04g0593200) are obtained, and / or

[0016] C. Place the plant gene Os04g0593200 under the regulation of a promoter with the nucleotide sequence SEQ ID NO:3 or a promoter with enhanced function.

[0017] The gene editing technologies mentioned above can be selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0018] A second aspect of the present invention provides a promoter for increasing the expression level of the transcription factor MYB41 (Os04g0593200), which is selected from:

[0019] (1) A polynucleotide with the nucleotide sequence SEQ ID NO:3;

[0020] (2) A polynucleotide whose nucleotide sequence is ≥95%, preferably ≥96%, preferably ≥97%, preferably ≥98%, more preferably ≥99% identical to the nucleotide sequence shown in SEQ ID NO:3, and which has the function of SEQ ID NO:1;

[0021] (3) A nucleotide sequence complementary to the nucleotide sequence described in (1) or (2).

[0022] A third aspect of the present invention provides a transcription factor MYB41 (Os04g0593200) gene expression cassette comprising the aforementioned promoter and the Os04g0593200 gene located downstream thereof.

[0023] A fourth aspect of the present invention provides a recombinant plasmid comprising the above-described transcription factor MYB41(Os04g0593200) gene expression cassette, and is suitable for expression in Agrobacterium.

[0024] A fifth aspect of the present invention provides a method for identifying rice varieties tolerant to salt stress, comprising the following steps:

[0025] Sequencing of the rice gene Os04g0593200, and / or

[0026] The determination was made to determine whether the protein expressed by the rice cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:1.

[0027] When the detection results show that the rice genome contains the gene MYB41 with the nucleotide sequence SEQ ID NO:2, or that the protein expressed by rice cells contains a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it indicates that the rice has a tendency to tolerate salt stress, and the rice variety is selected as a candidate for a rice variety tolerant to salt stress.

[0028] A sixth aspect of the present invention provides a kit for carrying out the above-described identification method, comprising the following PCR primers for amplifying the gene MYB41:

[0029] Forward primer RT-PCR-MYB41-F: ATGGGGAGGTCGCCGTGC (SEQ ID NO:5),

[0030] Reverse primer RT-PCR-MYB41-R: TTATGAACGGAAGCTGCTTTCT (SEQ ID NO:6).

[0031] Furthermore, the kit described above also includes the following PCR primers for detecting the internal reference gene Actin2:

[0032] Forward primer RT-PCR-Actin2-F: CATCTCTCAGCACATTCCAGCAG,

[0033] Reverse primer RT-PCR-Actin2-R: AGGAGGACGGCGATAACAGC.

[0034] Furthermore, the kit also includes an instruction manual, which describes the steps and identification criteria for detecting the rice gene Os04g0593200.

[0035] For example, the instructions can be written on bottles, test tubes and similar objects, boards, or on a separate piece of paper, or on the outside or inside of a container, such as a paper document with an operation demonstration video app download window or a QR code. The instructions can also be in multimedia form, such as a CD, USB flash drive, or cloud storage.

[0036] This invention is the first to discover that the transcription factor MYB41 (NCBI ID Os04g0593200) is associated with salt tolerance in rice and can maintain and improve the salt stress tolerance of rice. This function makes the gene MYB41 a valuable genetic resource that can be used to improve the salt stress tolerance of plants, especially rice, improve salt-tolerant plant varieties, and develop plant germplasm resources with high salt tolerance. Attached Figure Description

[0037] Figure 1 This image shows an analysis of the expression of the transcription factor MYB41 gene in the root epidermis of rice ZH11.

[0038] Figure 2 Comparative photographs of the growth phenotypes of wild-type rice ZH11 and the transcription factor MYB41 gene knockout mutant mybQ cultured in Yoshida nutrient solution. Figure 2 The mutant mybQ, resulting from the knockout of the transcription factor MYB41 gene Os04g0593200, exhibits dwarfism.

[0039] Figure 3 Comparative photographs of the growth phenotypes of wild-type rice ZH11 and the transcription factor MYB41 gene knockout mutant mybQ after being transplanted into 150 mmol NaCl Yoshida nutrient solution. Figure 3 The results showed that the mybQ mutant, resulting from the knockout of the transcription factor MYB41 gene Os04g0593200, exhibited stunted growth and was subjected to salt stress. Detailed Implementation

[0040] The synthesis of suberin molecules in rice is related to its resistance to salt sensitivity. Considering that MYB family transcription factors may be involved in regulating suberin molecule synthesis and cell wall suberin lamellar deposition, we studied and analyzed some transcription factors in the MYB family. We found that one of the transcription factors, MYB41 (NCBI code Os04g0593200), plays a positive role in improving the salt stress tolerance of rice. Its loss-of-function mutant plants are sensitive to salt and have poor growth phenotype, indicating that Os04g0593200 positively regulates the growth and salt tolerance of rice and also plays a role in maintaining normal plant growth.

[0041] We constructed a MYB41 gene knockout expression vector using CRISPR-Cas9 genetic engineering technology and transformed it into wild-type rice callus via Agrobacterium tumefaciens intrusion, resulting in the absence of MYB41 gene expression in the wild-type rice. The plants exhibited stunted growth and sensitivity to salt treatment. We then constructed a vector using the Os04g0593200 promoter to drive GUS (β-glucuronidase gene) expression, which was expressed in wild-type ZH11 rice. We observed its specific expression in the outer cortex of rice roots. To facilitate the identification and screening of transgenic plant cells or plants, the transformation vector contained antibiotic resistance markers (kanamycin, hygromycin).

[0042] Based on the above findings, this invention can use the gene MYB41 (Os04g0593200) as an indicator to identify salt-tolerant rice varieties. The identification method involves sequencing the rice gene Os04g0593200 and / or determining whether the protein expressed by the rice cells contains the polypeptide MYB41 with the amino acid sequence shown in SEQ ID NO:1. If the rice genome contains the gene MYB41 with the nucleotide sequence SEQ ID NO:2, and / or the protein expressed by the rice cells contains the polypeptide MYB41 with the amino acid sequence shown in SEQ ID NO:1, i.e., MYB41 is present and has not been mutated or inactivated, it indicates that the rice has a tendency to tolerate salt stress, and this rice variety can be considered a candidate for salt-tolerant varieties.

[0043] As used in this article, the term "wild-type" refers to native plants, such as rice, that have not undergone genetic modification or mutagenesis.

[0044] Correspondingly, the terms "(plant) mutant", "transgenic plant" and "genetically engineered plant" in this article have the same meaning, all referring to plants that have been genetically modified or artificially mutated from wild-type plants.

[0045] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0046] In this document, for the sake of simplicity, the name of a protein, such as MYB41, and its encoding gene (DNA), MYB41, are sometimes used interchangeably. Those skilled in the art should understand that they represent different types of substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or category of a transcription factor, MYB41 refers to a protein; when describing a gene, it refers to the gene encoding that protein.

[0047] On the other hand, given that the gene MYB41 with a normal sequence or its encoding transcription factor MYB41(Os04g0593200) has a positive effect on improving the salt stress tolerance of rice, it is possible to try to implement plant repair or salt-tolerant plant varieties by overexpressing the transcription factor MYB41(Os04g0593200) with the amino acid sequence shown in SEQ ID NO:1 in salt-sensitive plants such as rice varieties, thereby constructing the desired genetically engineered plants that are tolerant to salt stress.

[0048] The advantage of the aforementioned gene identification scheme lies in the ability to pre-assess the potential salt tolerance of candidate plant varieties solely in the laboratory. Since the entire life cycle of crops such as rice is typically one year or six months, examining their biological traits and phenotypes through field cultivation would normally require a significant amount of time and resources, incurring substantial land and labor costs. In contrast, the gene identification scheme can be completed in the laboratory, allowing for gene sequencing in a short period, such as on seedlings within a few weeks, or even just on seeds. This significantly improves efficiency and substantially reduces time, space, and labor costs, resulting in substantial economic benefits.

[0049] The construction of such salt-tolerant genetically engineered plants can be achieved using traditional Agrobacterium-mediated transformation with recombinant plasmids or gene editing technology.

[0050] In a specific implementation, the MYB41(Os04g0593200) expression cassette and nucleic acid construct or expression construct are constructed based on the gene MYB41 with nucleotide sequence SEQ ID NO:2, and then the MYB41 expression plasmid is constructed, and finally the MYB41 as a foreign gene is expressed in wild plants / primitives.

[0051] The coding sequence of the polypeptide MYB41 of the present invention, or a fragment thereof, can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, conventional techniques can be used to amplify the MYB41 gene from genomic DNA, and primers can be designed based on the nucleotide sequence disclosed in the present invention, especially the open reading frame sequence.

[0052] As used herein, the terms "expression cassette," "gene expression cassette," or "nucleic acid construct" refer to a gene expression system containing all the necessary elements required to express the target polypeptide MYB41. Typically, it includes the following elements: a promoter, a gene sequence encoding the polypeptide, and a terminator; optionally, it may also include a signal peptide coding sequence, etc.; these elements are operatively linked. In this invention, the preferred promoter for regulating MYB41 gene expression is a polynucleotide SEQ ID NO:3.

[0053] As used herein, the term "expression construct" or "expression building block" refers to a recombinant DNA molecule containing the intended nucleic acid coding sequence SEQ ID NO:2, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector (plasmid vector).

[0054] As used herein, “operationally linked” or “operationally connected” refers to a functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence SEQ ID NO:2, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0055] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the polypeptide or transcription factor MYB41 (Os04g0593200). The nucleic acid constructs can be manipulated according to the expression vector or requirements before insertion into the vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0056] In some embodiments, the nucleic acid construct is a vector. The vector can be a cloning vector, an expression vector, or a gene knock-in vector. The nucleic acid sequence SEQ ID NO:2 of the present invention can be cloned into many types of vectors, such as plasmids, phage particles, phage derivatives, animal viruses, and granules. Cloning vectors can be used to provide the coding sequence of the protein or polypeptide of the present invention. Expression vectors can be provided to cells in the form of bacterial or viral vectors. Expression of the MYB41 gene of the present invention is typically achieved by operably linking the nucleic acid sequence SEQ ID NO:2 of the present invention to the promoter SEQ ID NO:3 and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. A typical expression vector contains expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0057] Gene knock-in vectors can be used to integrate the polynucleotide sequence SEQ ID NO:2 described herein into a region of interest in the host genome. Typically, gene knock-in vectors contain the polynucleotide sequence described herein, as well as 5' and 3' homologous arms required for genomic homologous recombination. In some embodiments, the nucleic acid constructs described herein contain 5' homologous arms, the polynucleotide sequence described herein, and 3' homologous arms. When using gene knock-in vectors, CRISPR / Cas9 technology can be used simultaneously to homologously recombine the polynucleotide sequence into the site of interest. CRISPR / Cas9 technology guides the Cas9 nuclease to modify the genome at the insertion site by designing guide RNAs targeting the target gene, resulting in increased homologous recombination efficiency in the modified region, thus homologously recombinating the target fragment SEQ ID NO:2 contained in the gene knock-in vector into the target site. The steps of CRISPR / Cas9 technology and the reagents used, such as the Cas9 nuclease, are well known in the art.

[0058] Methods well known to those skilled in the art can be used to construct nucleic acid constructs. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be efficiently ligated to an appropriate promoter in the expression vector to direct mRNA synthesis. Representative examples of these promoters include: the lac or trp promoter of *E. coli*; the PL promoter of *λ* phage; eukaryotic promoters including the CMV immediate early promoter, the HSV thymidine kinase promoter, early and late SV40 promoters, LTRs of retroviruses, and other known promoters that control gene expression in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Furthermore, the expression vector preferably contains one or more selective marker genes to provide phenotypic traits for selecting host cells for transformation, such as dihydrofolate reductase, neomycin resistance, and green fluorescent protein (GFP) for eukaryotic cell culture, or tetracycline, ampicillin resistance, or chloramphenicol for *E. coli*, *Agrobacterium*, etc.

[0059] When the polynucleotides of this invention are expressed in higher eukaryotic cells, the insertion of an enhancer sequence into the vector will enhance transcription. Enhancers are cis-acting factors of DNA, typically approximately 10 to 300 base pairs, that act on the promoter to enhance gene transcription. Examples include the SV40 enhancer (100 to 270 base pairs) located late on the replication origin side, the polyoma enhancer located late on the replication origin side, and adenovirus enhancers.

[0060] Vectors containing appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

[0061] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0062] 1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the coding sequence of the polypeptide MYB41;

[0063] 2) Contact the plant cells, tissues or organs with the Agrobacterium in step (1) to transfer the coding sequence into the plant cells and integrate it into the chromosomes of the plant cells;

[0064] 3) Select plant cells or tissues into which the coding sequence has been introduced; and

[0065] 4) Regenerate plants from the plant cells or tissues in step 3).

[0066] The method described herein can be used to construct transgenic plants with different uses, such as transgenic plants for environmental remediation and crops tolerant to salt stress.

[0067] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0068] Example

[0069] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0070] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0071] The molecular biology experiments in this embodiment, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were both performed according to Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments if necessary.

[0072] PCR amplification experiments should be performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0073] The primer synthesis and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0074] The Os04g0593200 knockout mutant mybQ and the transgenic rice with GUS expression driven by the Os04g0593200 promoter were both constructed and cultivated by Weimi Biotechnology (Changzhou) Co., Ltd.

[0075] Example 1: Cloning of gene Os04g0593200

[0076] Based on various rice varieties sensitive to salt stress and salt-tolerant rice varieties, our research group previously discovered and cloned the gene Os04g0593200, which is related to rice salt tolerance, through transcriptome sequencing comparison. The nucleotide sequence of this gene is SEQ ID NO:2, which encodes a transcription factor MYB41 (abbreviated as transcription factor MYB41, or OsMYB41) with an amino acid sequence as shown in SEQ ID NO:1.

[0077] Example 2: Construction of the Os04g0593200 knockout mutant mybQ

[0078] Using wild-type rice ZH11 (Zhonghua 11) as the wild type, Weimi Biotechnology (Changzhou) Co., Ltd. was commissioned to construct and cultivate a gene Os04g0593200 knockout mutant, which is a mutant in which the gene Os04g0593200 has been knocked out in the chromosome genome, and named mybQ.

[0079] The mutant plant mybQ was transplanted to the Songjiang Base of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, for field cultivation to obtain seeds of the mutant mybQ.

[0080] Example 3: Construction of transgenic rice with GUS expression driven by the Os04g0593200 gene promoter

[0081] Using wild-type rice ZH11 (Zhonghua 11) as the wild type, Weimi Biotechnology (Changzhou) Co., Ltd. was commissioned to construct and cultivate a transgenic line expressing the reporter gene GUS. Its chromosomal genome contains the promoter Os04g0593200 SEQ ID NO:3 and the downstream GUS encoding gene.

[0082] The transgenic seedlings were transplanted to the Songjiang Base of the Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, for field cultivation and subsequent transgenic identification.

[0083] Example 4: Investigating the expression distribution of gene Os04g0593200 in wild-type rice

[0084] Taking the expression of the Os04g0593200 gene in roots as an example, the following steps are included.

[0085] 1. Samples were taken from ZH11 transgenic seedlings whose GUS (β-glucuronidase gene) expression was driven by the promoter SEQ ID NO:3 of the transcription factor MYB41 (NCBI number Os04g0593200) gene. A 2 mm section was cut from 3.5 cm away from the root tip, embedded in 0.5% agarose, trimmed, and sectioned into 50 μm thick sections using a vibratory microtome.

[0086] 2. Preparation of GUS staining solution: Prepare a 20 mM stock solution of X-Gluc (5-bromo-4-chloro-3-indole-β-D-glucuronide cyclohexylamine salt) powder using DFM. Dilute with GUS buffer to a final concentration of 1 mM working solution. The GUS buffer formula is as follows: 50 mM sodium dihydrogen phosphate, 50 mM disodium hydrogen phosphate, 1 mM GUS, 10 mM EDTA, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, 0.1% Triton X-100, and adjust the pH to 8.0.

[0087] 3. Place the sections in GUS staining solution, evacuate the vacuum pump for 30 minutes, and continue staining at 37 degrees Celsius in the dark for 5 hours. After staining, discard the staining solution, wash three times to remove excess stain, and observe under an optical microscope. The results are shown in the figure. Figure 1 . Figure 1 The transcription factor MYB41 gene was found to be expressed in the outer cortex of the root in rice ZH11.

[0088] Example 5: Investigating the effect of gene knockout Os04g0593200 on rice growth

[0089] 1. Soak the mutant mybQ seeds and wild-type ZH11 seeds in tap water and place them in a 37℃ oven for germination. After germination for two days, place them in a 96-well plate with the bottom removed and culture them in tap water.

[0090] 2. After culturing in tap water for three days, the mutant mybQ and wild-type ZH11 seedlings were transferred to a nutrient solution for one week of cultivation. The nutrient solution formula is as follows:

[0091] The 1000X Yoshida mother liquors are as follows:

[0092] Mother liquor A: NH4NO3 80g / L

[0093] NaH2PO4·2H2O 93g / L

[0094] K2SO4 52.4g / L

[0095] Mother liquor B: CaCl2·2H2O 44.2g / L

[0096] Mother liquor C: MgCl2·6H2O 122g / L

[0097] Mother liquor D: Fe-EDTA 19 g / L

[0098] Mother liquor E: H3BO3 3.01g / L

[0099] MnSO4·5H2O 2.17g / L

[0100] CuSO4·5H2O 0.075g / L

[0101] ZnSO4·7H2O 0.2008g / L

[0102] Na2M O O4·2H2O 0.024g / L

[0103] 3. The growth phenotype of rice seedlings after 14 days of growth is shown in [the figure]. Figure 2 . Figure 2The photos show that the mutant mybQ plants are shorter than the wild type, suggesting that the transcription factor MYB41 is important for maintaining normal growth of rice plants.

[0104] Example 6: Comparison of salt stress tolerance between mutant mybQ and wild-type ZH11

[0105] Rice seedlings that had grown for 14 days were transplanted into a Yoshida nutrient solution containing 150 mM NaCl and cultured in an incubator for observation. The results are shown below. Figure 3 . Figure 3 The results indicate that the mybQ mutant plant, after the Os04g0593200 gene was knocked out, exhibited a salt-sensitive phenotype and its growth was affected by salt stress.

[0106] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. Application of transcription factor MYB41 (NCBI number Os04g0593200) with amino acid sequence as shown in SEQ ID NO:1 in improving plant salt tolerance.

2. The application as described in claim 1, characterized in that, The plant is a grass crop, selected from rice, wheat, corn, soybean, barley, oats, rye and sorghum, with rice being the preferred crop.

3. The application as described in claim 1, characterized in that, The nucleotide sequence of the gene expressing the transcription factor MYB41 is SEQ ID NO:

2.

4. The application as described in claim 1, characterized in that, Used to create salt-tolerant plant germplasm or to cultivate salt-tolerant plant varieties.

5. The application as described in claim 4, characterized in that, Salt-tolerant plant germplasm can be created or salt-tolerant plant varieties can be cultivated by overexpressing the transcription factor MYB41 or its expression gene MYB41 in plants.

6. The application as described in claim 5, characterized in that, The overexpression of the transcription factor MYB41 (Os04g0593200) or its expression gene MYB41 is achieved through the following method: A. The gene MYB41 with the nucleotide sequence SEQ ID NO:2 as described in claim 3 is cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely the MYB41 overexpression vector. Plants are then transformed by Agrobacterium-mediated transformation to obtain transgenic plants that overexpress the transcription factor MYB41 (Os04g0593200). B. By using gene editing technology, the gene MYB41 with the nucleotide sequence SEQ ID NO:2 as described in claim 3 is cloned into a plant chromosome to obtain a transgenic plant overexpressing the transcription factor MYB41 (Os04g0593200); and / or C. Place the plant gene Os04g0593200 under the regulation of a promoter with the nucleotide sequence SEQ ID NO:3 or a promoter with enhanced function.

7. A promoter for increasing the expression level of the transcription factor MYB41 (Os04g0593200) as described in claim 1, characterized in that, It is selected from: (1) A polynucleotide with the nucleotide sequence SEQ ID NO:3; (2) A polynucleotide whose nucleotide sequence is ≥95% identical to the nucleotide sequence shown in SEQ ID NO:3, and which has the function of SEQ ID NO:1; (3) A nucleotide sequence complementary to the nucleotide sequence described in (1) or (2).

8. A transcription factor MYB41 (Os04g0593200) gene expression cassette, characterized in that, It comprises the promoter as described in claim 7 and the Os04g0593200 gene located downstream therefrom; and A recombinant plasmid, characterized in that it contains the above-mentioned transcription factor MYB41(Os04g0593200) gene expression cassette and is suitable for expression in Agrobacterium.

9. A method for identifying rice varieties tolerant to salt stress, characterized in that, Includes the following steps: Sequencing of the rice gene Os04g0593200, and / or The determination was made to determine whether the protein expressed by the rice cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:

1. When the detection results show that the rice genome contains the gene MYB41 with the nucleotide sequence SEQ ID NO:2, or that the protein expressed by rice cells contains a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it indicates that the rice has a tendency to tolerate salt stress, and the rice variety is selected as a candidate for a rice variety tolerant to salt stress. When the rice genome does not contain the gene MYB41 with the nucleotide sequence SEQ ID NO:2, or when the protein expressed by rice cells does not contain the polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that rice is at risk of salt intolerance.

10. A kit for carrying out the method as described in claim 9, characterized in that, The following PCR primers are included for amplifying the MYB41 gene: Forward primer RT-PCR-MYB41-F: ATGGGGAGGTCGCCGTGC (SEQ ID NO:5), Reverse primer RT-PCR-MYB41-R: TTATGAACGGAAGCTGCTTTCT (SEQ ID NO:6).

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