Glycerol acyltransferase affecting rice keratin synthesis

Abstract

The invention belongs to the technical field of agricultural biology, and relates to application of glycerol acyltransferase with an amino acid sequence as shown in SEQ ID NO: 1 to improvement of salt tolerance of plants, and the glycerol acyltransferase is of great significance to creation of salt-tolerant plant germplasm resources and cultivation of salt-tolerant rice varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a glycerol acyltransferase that affects cutin synthesis in rice (issued as Os01g0855000 on the rice website https: / / rapdb.dna.affrc.go.jp / ; NCBI number LOC4324826) 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, a comprehensive understanding of how 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. Many articles have reported that suberization occurs prematurely in plant roots after salt treatment. Suberization refers to the deposition of suberin in the cell wall, with the entire cell enveloped by suberin. Suberin acts as an apoplastic "barrier" in the endoderm and epidermis of plant roots, playing a role in ultrafiltration of sodium ions. Suberin, as a "barrier," has been shown to potentially participate in the plant's salt tolerance process (Franke R, Suberin—a biopolyester forming apoplastic plant interfaces. Curr Opin Plant Biol. 2007.).

[0003] Suberin is a substance composed of glycerol esters and phenols as basic units. Existing research shows that glycerol-3-phosphate acyltransferases (GPATs) are involved in the synthesis of suberin in roots. GPATs catalyze the first step in glycerol ester synthesis, mainly participating in the synthesis of epidermal lipids, membrane lipids, and oils. It catalyzes the acylation reaction at the sn-1 and sn-2 positions of glycerol-3-phosphate (G3P) to form lysophosphatidic acid (LPA). Then, under the catalysis of LPA acyltransferase (LPAAT), LPA is dephosphorylated to form phosphatidic acid (PA), thus participating in the de novo synthesis of glycerol lipids. In the glycerol lipid synthesis pathway, the synthesis of phosphatidic acid in plants is divided into two categories: the prokaryotic pathway and the eukaryotic pathway. The prokaryotic pathway mainly takes place in chloroplasts, while the eukaryotic pathway takes place in the endoplasmic reticulum. Chloroplast-localized GPAT, also known as AISI. Most members of the GPAT family, namely GPAT1-GPAT8, can utilize α-oxidized acyl-CoA or ultra-long-chain acyl-CoA as acyl donors to acylate at the sn-2 position of G3P, forming sn-2LPA or sn-2MAG (sn-2monoacylglycerol 2-MAG), and participate in the synthesis of extracellular matrix such as cuticle and cork.

[0004] In Arabidopsis thaliana, it has been reported that GPAT5 and GPAT7 are involved in the synthesis of suberin in the roots, while GPAT6 is mainly expressed in the flowers, causing pollen wall adhesion and affecting fertility.

[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 discovered a new glycerol-3-phosphate 2-O-acyltransferase 6 (GLP-6, identified as Os01g0855000 in the rice website https: / / rapdb.dna.affrc.go.jp / and NCBI ID LOC4324826, referred to as "glycerol acyltransferase" or GPAT2O6 or OsGPAT6 in this paper) that can affect rice cuticle synthesis. Loss-of-function mutants of this enzyme exhibited salt sensitivity and poorer phenotypes, indicating that Os01g0855000 positively regulates rice salt tolerance. Furthermore, we discovered a promoter that efficiently regulates the expression of the Os01g0855000 gene, promoting its expression in rice roots and leaves, and high expression in the pollen wall. Based on these findings, this invention includes the following technical solutions.

[0007] The first aspect of this invention provides the application of a glycerol acyltransferase (Os01g0855000) with the amino acid sequence shown in SEQ ID NO:1 or its expression gene GPAT2O6 in improving plant salt tolerance:

[0008] MVSRRFKPVEECSSDGRSEQTVAADFDGTLVRSRSAFPYYLLVALEAGSVLRAVVLLLSVPFVYVTYIFFSESLAISTLVYISVAGLKVRNIEMVARSVLPKFYAEDVHPESWRVFNSFGKRYII TASPRIMVEHFAKTFLGADKVVGTELEVGKNGKATGFMVKPGVLVGDHKRQAVVKELRDAVPDVGLGDRETDFDFMSICKEAYLVTSRKYSAVPKNQLLSPLILHDGRLVQRPTPLVALVTFLWM PFGFALALLRVYVNLPLPERIVFYTYKLMGIRLIVKGNPPPPPKKGHPGVLFVCNHRTVLDPVEVAVALRRKVSCVTYSISKFSELISPIKAVALSREREKDAENIRRLLEEGDLVICPEGTTCR EPFLLRFSALFAELTDRIVPVAINTKESMFHGSTVRGFKLMDPYFFFMNPRPTYEITFLNQLPKELTCSGGKSPIEVANYIQKTLSGQLGFECTAITRKEKYSILAGTDGRVPSKNKEKEKN(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 GPAT2O6 of the above-mentioned glycerol acyltransferase GPAT2O6 (Os01g0855000) is SEQ ID NO:2, which consists of a 5'UTR region, a 3'UTR region, introns and exons (coding region, i.e., CDS).

[0012] In another embodiment, the nucleotide sequence of the expression gene GPAT2O6 of the above-mentioned glycerol acyltransferase GPAT2O6 is SEQ ID NO:3, which is the coding region, i.e., the CDS sequence, in SEQ ID NO:2.

[0013] In the above-mentioned application methods, salt-tolerant plant germplasm is created or salt-tolerant plant varieties are cultivated by overexpressing glycerol acyltransferase (Os01g0855000) or its expression gene GPAT2O6 in plants.

[0014] In the above application method, the overexpression of glycerol acyltransferase (Os01g0855000) or its expression gene GPAT2O6 is achieved through the following method:

[0015] A. The gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 was cloned into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely the GPAT2O6 overexpression vector. Plants were then transformed using Agrobacterium-mediated transformation to obtain transgenic plants overexpressing glycerol acyltransferase (Os01g0855000); or

[0016] B. By using gene editing technology, the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 was cloned into the plant chromosome to obtain transgenic plants that overexpress glycerol acyltransferase (Os01g0855000).

[0017] C. Place the plant gene Os01g0855000 under the regulation of a promoter with the nucleotide sequence SEQ ID NO:4 or a promoter with enhanced function.

[0018] 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.

[0019] A second aspect of the present invention provides a promoter for increasing the expression level of the above-mentioned glycerol acyltransferase (Os01g0855000), which is selected from:

[0020] (1) A polynucleotide with the nucleotide sequence SEQ ID NO:4;

[0021] (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:4, and the polynucleotide has the function of SEQ ID NO:1, which means driving the expression of the Os01g0855000 gene in rice roots, leaves and pollen walls;

[0022] (3) A nucleotide sequence complementary to the nucleotide sequence described in (1) or (2).

[0023] A third aspect of the present invention provides a glycerol acyltransferase (Os01g0855000) gene expression cassette comprising the promoter described above and the Os01g0855000 gene located downstream thereof.

[0024] A fourth aspect of the present invention provides a recombinant plasmid comprising the above-described glycerol acyltransferase (Os01g0855000) gene expression cassette, and is suitable for expression in Agrobacterium.

[0025] A fifth aspect of the present invention provides a method for identifying rice varieties tolerant to salt stress, comprising the following steps:

[0026] Sequencing of the rice gene Os01g0855000, and / or

[0027] 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.

[0028] When the detection results show that the rice genome contains the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when 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 salt-tolerant rice varieties.

[0029] When the rice genome does not contain the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the protein expressed by rice cells does not contain a polypeptide with the amino acid sequence shown in SEQ ID NO:1, it suggests that rice is at risk of salt intolerance.

[0030] A sixth aspect of the present invention provides a kit for performing the above-described identification method, comprising the following PCR primers for amplifying the gene GPAT2O6:

[0031] Forward primer RT-PCR-GPAT2O6-F: CGTTCGTCTACGTGACCTACAT (SEQ ID NO:5),

[0032] Reverse primer RT-PCR-GPAT2O6-R: GAGGTAGGCCTCCTTGCATA (SEQ ID NO:6).

[0033] Furthermore, the kit described above also includes the following PCR primers for detecting the internal reference gene Actin2:

[0034] Forward primer RT-PCR-Actin2-F: AAGATGGCTGACGCCGAGGATATC,

[0035] Reverse primer RT-PCR-Actin2-R: AACTTCGATGTTATTCATTTCATA.

[0036] Furthermore, the kit also includes an instruction manual, which describes the steps and identification criteria for detecting the rice gene Os01g0855000.

[0037] 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.

[0038] This invention is the first to discover that the glycerol acyltransferase GPAT2O6 (Os01g0855000) affects cuticle synthesis in rice, thereby resisting salt stress and conferring salt tolerance to rice. This function makes the GPAT2O6 gene a valuable gene resource that can be used to improve the salt stress tolerance of plants, especially rice, to improve salt-tolerant plant varieties and develop highly salt-tolerant plant germplasm resources. In addition, the promoter-regulated gene Os01g0855000 with the nucleotide sequence SEQ ID NO:4 is expressed in rice roots and leaves, and highly expressed in pollen walls. Attached Figure Description

[0039] Figure 1 The images show the expression analysis of the Os01g0855000 gene in the aerial parts and roots of rice ZH11. The left image shows the pollen wall, the middle image shows the guard cells of the leaf stomata, and the right image shows the endodermal cells of the root. Figure 1This indicates that the Os01g0855000 gene is expressed in both the roots and leaves of rice ZH11.

[0040] Figure 2 Phenotypic images of the Os01g0855000 transgenic knockout material at the seedling and mature stages. The left image shows wild-type ZH11 and the mutant gpat6 seedlings, while the right image shows wild-type ZH11 and the mutant gpat6 seedlings at the tillering stage. Figure 2 This indicates that the mutant gpat6, resulting from the knockout of the Os01g0855000 gene, exhibits dwarfism.

[0041] Figure 3 Photographs of leaves stained with toluidine blue for wild-type ZH11 and Os01g0855000 gene knockout materials. Figure 3 The mutant gpat6, resulting from the knockout of the Os01g0855000 gene, exhibits leaf cuticle defects and increased permeability.

[0042] Figure 4 These are photographs of gpat6 plants, a mutant resulting from the knockout of the wild-type ZH11 and Os01g0855000 genes. The three plants on the left are the gpat6 mutant, and the three plants on the right are the wild-type ZH11. Figure 4 This indicates that the Os01g0855000 gene knockout material exhibits a salt-sensitive phenotype and its growth is affected by salt stress. Detailed Implementation

[0043] For the first time, we have discovered a relationship between the expression level of the rice genome gene Os01g0855000, which reduces cuticle synthesis in plant leaves, and rice salt stress tolerance. This gene, identified in the Rice Database (https: / / rapdb.dna.affrc.go.jp / index.html), exhibits a loss of function that leads to increased salt sensitivity and a worsened phenotype, indicating that Os01g0855000 positively regulates rice salt stress tolerance.

[0044] According to the genome sequence published in the Rice Database, Os01g0855000 is a glycerol-3-phosphoacyltransferase gene that has been extensively studied in plants in recent years. We found that Os01g0855000 gene loss-of-function mutants exhibited leaf cuticle defects and increased permeability; simultaneously, salt tolerance phenotype analysis of Os01g0855000 gene knockout plants revealed that they were sensitive to salt. Furthermore, this gene is expressed in pollen, and knocking out Os01g0855000 significantly reduces rice yield.

[0045] A CRISPR-CAS9 knockout expression vector was constructed using genetic engineering techniques and transformed into wild-type rice callus via Agrobacterium tumefaciens intrusion, resulting in the absence of the Os01g0855000 gene and stunted plant growth. We then constructed a vector using the Os01g0855000 promoter (nucleotide sequence SEQ ID NO:4) to drive the expression of the reporter gene GUS (β-glucuronidase gene), enabling GUS expression in wild-type rice. Expression was observed in the endodermis of rice roots, in leaf stomata, and at high levels in pollen walls. To facilitate the identification and screening of transgenic plant cells or plants, the transformation vector contained antibiotic resistance markers (kanamycin, hygromycin).

[0046] The study also found that the promoter-regulated gene Os01g0855000 with the nucleotide sequence SEQ ID NO:4 is expressed in the endodermis of rice roots, in leaf stomata, and highly expressed in pollen walls. This can help promote the function of the gene Os01g0855000, thereby improving the salt stress tolerance of plants, especially rice, and other excellent properties related to reducing / decreasing cuticle synthesis in plant leaves.

[0047] Based on the above findings, this invention can use the gene GPAT2O6 (Os01g0855000) as an indicator to identify salt-tolerant rice varieties. The identification method involves sequencing the rice gene Os01g0855000 and / or determining whether the protein expressed by the rice cells contains the polypeptide GPAT2O6 with the amino acid sequence shown in SEQ ID NO:1. If the rice genome contains the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, and / or the protein expressed by the rice cells contains the polypeptide GPAT2O6 with the amino acid sequence shown in SEQ ID NO:1, i.e., GPAT2O6 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.

[0048] As used in this article, the term "wild-type" refers to native plants, such as rice, that have not undergone genetic modification or mutagenesis.

[0049] 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.

[0050] 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).

[0051] As used herein, the terms “(plant leaf cutin synthesis) reduction,” “decline,” or “reduction” can mean a reduction of at least 10% relative to a reference level (e.g., wild-type rice / initial rice), such as a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any reduction between 10% and 100%, or a reduction of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times relative to a reference level.

[0052] In this document, for the sake of simplicity, the name of a protein, such as GPAT2O6, and its encoding gene (DNA), GPAT2O6, 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 reducing cuticle synthesis in plant leaves, GPAT2O6 refers to a protein; when describing it as a gene, it refers to the gene encoding that protein.

[0053] On the other hand, given that the gene GPAT2O6 with a normal sequence or its encoded glycerol acyltransferase (Os01g0855000) has a positive effect on reducing cuticle synthesis in plant leaves and improving the salt stress tolerance of rice, it is possible to try to implement plant repair or salt-tolerant plant varieties by overexpressing the glycerol acyltransferase (Os01g0855000) 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.

[0054] 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.

[0055] The construction of such salt-tolerant genetically engineered plants can be achieved using traditional Agrobacterium-mediated transformation with recombinant plasmids or gene editing technology.

[0056] In a specific implementation, the GPAT2O6(Os01g0855000) expression cassette and nucleic acid construct or expression construct are constructed based on the gene GPAT2O6 with nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, and then the GPAT2O6 expression plasmid is constructed. Finally, GPAT2O6 as a foreign gene is expressed in wild plants / primitives.

[0057] The coding sequence or fragment thereof of the polypeptide GPAT2O6 of the present invention can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, conventional techniques can be used to amplify the GPAT2O6 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.

[0058] 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 for expressing the target polypeptide GPAT2O6. 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 encoding sequence, etc.; these elements are operatively linked. In this invention, the preferred promoter for regulating GPAT2O6 gene expression is polynucleotide SEQ ID NO:4.

[0059] As used herein, an "expression construct" or "expression building block" refers to a recombinant DNA molecule containing the intended nucleic acid coding sequence SEQ ID NO:2 or 3, which may contain one or more gene expression cassettes. The "construct" is typically contained within an expression vector (plasmid vector).

[0060] 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 or 3, 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.

[0061] The nucleic acid constructs described in this invention can be manipulated in various ways to ensure the expression of the polypeptide or glycerol acyltransferase (Os01g0855000). 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.

[0062] 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 or 3 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 GPAT2O6 gene of the present invention is typically achieved by operably linking the nucleic acid sequence SEQ ID NO:2 or 3 of the present invention to a promoter and incorporating the construct into an expression vector. This vector is suitable for replication and integration into eukaryotic cells. Typical expression vectors contain expression control sequences that can be used to regulate the expression of the desired nucleic acid sequence.

[0063] Gene knock-in vectors can be used to integrate the polynucleotide sequence SEQ ID NO:2 or 3 described herein into a region of interest in the host genome. Typically, in addition to the polynucleotide sequence described herein, gene knock-in vectors may also contain 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 or 3 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] When constructing transgenic plants using the traditional Agrobacterium-mediated transformation method, the methods for constructing transgenic plants include:

[0068] 1) Provide Agrobacterium carrying an expression vector, wherein the expression vector contains the coding sequence of the polypeptide GPAT2O6;

[0069] 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;

[0070] 3) Select plant cells or tissues into which the coding sequence has been introduced; and

[0071] 4) Regenerate plants from the plant cells or tissues in step 3).

[0072] 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.

[0073] 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.

[0074] Example

[0075] 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.

[0076] In the embodiments described herein, unless otherwise specified, the temperature generally refers to room temperature (15-30°C).

[0077] 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.

[0078] 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.

[0079] The primer synthesis and gene sequencing in this embodiment were outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0080] Example 1: Construction of the Os01g0855000 gene knockout mutant gpat6

[0081] The wild-type rice material Zhonghua 11 (Oryza sativa L. subsp. japonicacv. Zhonghua 11) used in this paper was bred and preserved in our laboratory. Field phenotypic identification, photography, and sample collection for genotyping of all rice materials, including transgenic materials, were all completed in Shanghai. The construction of the Os01g0855000 gene knockout mutant gpat6 included the following steps.

[0082] 1. We used the Huazhong Agricultural University CRISPR-P website for design ( http: / / crispr.hzau.edu.cn / CRISPR2 / The gene target was used to create rice gene knockout mutant materials. The plasmid construction in the CRISPR / Cas system was handled by Baige Gene Technology Co., Ltd., and the knockout target was GTGAAGCCCGGAGTGCTCGTGGG. The PCR system and process are as follows.

[0083] Prepare a 50 μL system and carry out the amplification reaction according to the following procedure.

[0084] PCR system:

[0085]

[0086] PCR procedure:

[0087]

[0088]

[0089] Using 1.5% agarose gel electrophoresis at 5 V / cm for 20 minutes, the t1-t2 (203 bp) electrophoretic fragment was excised under UV light and placed in a system for sol-gel recovery. The recovery procedure is as described in the Novizan kit instructions. The recovered DNA was dissolved in 30 μL of water (the recovered product was labeled as: rDNAt1). After verification, it was ligated into the vector.

[0090] 2. Enzyme digestion and ligation

[0091] Enzyme digestion and ligation system:

[0092]

[0093] Enzyme digestion and ligation reaction conditions:

[0094]

[0095] The ligation product was transformed into competent cells.

[0096] 3. Transformation

[0097] Transform 5-10 μL of the ligation product into competent E. coli cells. Plate the transformants onto kanamycin-resistant plates and incubate at 37°C for 12 hours. Perform plasmid PCR identification. Extract plasmids from correctly identified E. coli cells for later use.

[0098] 4. Agrobacterium-mediated genetic transformation of rice. The specific steps are as follows:

[0099] (1) Rice callus induction: Plump, hulled rice seeds were washed with 75% (v / v) ethanol for 1 min, then soaked in 2.5% (v / v) sodium hypochlorite for 45 min. After washing three times with sterile water, the seeds were sown on NB medium. Callus tissue grew at the mature embryo scutellum after about 15 days. Smooth, dense, and pale yellow embryogenic callus tissue was selected and subcultured on NB medium, and then subcultured every 14 days. The NB medium (also called NB basal medium) consists of: 2830 mg / L KNO3, 463 mg / L (NH4)2SO4, 400 mg / L KH2PO4, 185 mg / L MgSO4·7H2O, 166 mg / L CaCl2·2H2O, 27.8 mg / L FeSO4·7H2O, 37.5 mg / L Na2EDTA, 10 mg / L MnSO4·4H2O, 3 mg / L H3BO3, 2 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 0.75 mg / L KI, 10 mg / L Vitamin B1, 1 mg / L Vitamin B6, and 1 mg / L Nicotinic acid. acid, 100mg / LMyo-inositol, 300mg / L Casein hydrolysate, 500mg / Llutamine, 2mg / L Glycine, 1000mg / L Proline, 2mg / L 2,4-D.

[0100] (2) Agrobacterium transformation and culture: 1-2 μL of the successfully constructed transgenic plasmid was added to Agrobacterium competent cells EHA105. After electroporation, the cells were cultured at 28°C for 1 h. Then, the plasmid was evenly spread onto YEP solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and incubated upside down at 28°C for 2 days. Single Agrobacterium colonies were picked and placed in 1 mL of YEP liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and cultured at 28°C for 9 h. The colonies were then transferred to 50 mL of YEP liquid medium containing 50 mg / L kanamycin, 25 mg / L rifampin, and 19.6 mg / L acetylsylphenone. After overnight culture at 28°C for 12 h, the bacterial pellet was collected by centrifugation at 3,000 rpm for 10 min and resuspended in inoculum. The YEP medium consisted of 10 g / L yeast extract, 10 g / L Bacto Peptones, and 5 g / L NaCl. YEP solid medium: Based on the above components, add 15 g / L agar and sterilize at 120℃ for 20 min. The composition of the infiltration solution is: 1×NB basal medium, 2 g / L Inositol, 2 g / L Glutamine, 500 mg / L Casein hydrolysate, 10 mL / L 10% (w / v) Synperonic PE, and 100 μM Cetosyringone.

[0101] (3) Agrobacterium infection: Rice callus cultured on fresh NB medium for 4 days was transferred to a 100 mL sterile Erlenmeyer flask, and the resuspended Agrobacterium infection solution was added. The flask was incubated at room temperature for 20 min, with shaking every 5 min. The rice callus was removed and excess bacterial solution was blotted off with sterile filter paper. The flask was then placed on NB solid medium containing 100 μM acetylsyl syringone and covered with a layer of sterile filter paper, and incubated at 26 °C in the dark for 2-3 days.

[0102] (4) Screening, differentiation, and plant regeneration of resistant callus: Agrobacterium-infected rice callus was transferred to NB medium containing 50 mg / L hygromycin and cultured for 7 days. Afterward, it was transferred to a new selection medium for the next round of screening. This screening process was repeated four times. The resulting resistant callus was then placed on differentiation medium for approximately 30 days of differentiation. The differentiated rice seedlings had their roots removed and were placed on rooting medium for secondary rooting culture. When the rice seedlings reached approximately 15 cm in height, they were hardened off for 7 days before being transplanted to the experimental field.

[0103] After the above steps, a mutant with the gene Os01g0855000 knocked out in the chromosome genome of wild-type rice ZH11 (Zhonghua 11) was obtained and named gpat6.

[0104] The mutant plant gpat6 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 gpat6.

[0105] Example 2: Construction of transgenic rice with GUS expression driven by the Os01g0855000 gene promoter

[0106] 1. Construct the pGPAT-GUS-P1300 plasmid.

[0107] First, synthesize the following primer pair:

[0108] pOs01g0855000-GUS-1300-F:

[0109] GTAAAACGACGGCCAGTGCCAAGCTTACAGTGAGTGCGTAACTCCTG

[0110] pOs01g0855000-GUS-1300-R:

[0111] TTTACCCTCAGATCTACCATGGTACCCTGCTCCGACCTCCCATCCG.

[0112] PCR using rice genome as a template

[0113] Prepare a 50 μL system and carry out the amplification reaction according to the following procedure.

[0114] PCR system

[0115]

[0116] PCR program

[0117]

[0118] The 2792bp fragment was extracted by 1% agarose gel electrophoresis at 5V / cm for 20 minutes under UV light and placed in a system for sol-gel recovery. The recovery procedure is as described in the Novizan kit instructions. The recovered DNA was dissolved in 30μL of water and, after verification, ligated into the vector.

[0119] 2. Enzyme digestion and ligation

[0120] Enzyme ligation system and reaction conditions

[0121]

[0122] React at 37℃ for 2 hours.

[0123] Homologous recombination system and reaction conditions:

[0124]

[0125] React at 37℃ for 30 minutes

[0126] The ligation product was transformed into competent cells.

[0127] 3. Transformation

[0128] Transform 5-10 μL of the ligation product into competent E. coli transformants, plate them on kanamycin-resistant plates, and incubate at 37°C for 12 hours. Perform plasmid PCR identification. Extract plasmids from correctly identified E. coli strains for later use.

[0129] The plasmid was then transformed into Agrobacterium, as described in Example 2.

[0130] Rice callus that had completed the co-culture stage was transferred to a selective medium containing 50 mg / L hygromycin and 100 mg / L carbenicillin for the first selection culture. Seven days later, the surviving callus was transferred to a selective medium containing 50 mg / L hygromycin and 50 mg / L kanamycin for the second selection. Selection was then performed every 7 days for a total of four selections. Vigorous resistant callus was then selected and transferred to differentiation culture for a 30-40 day differentiation period. The differentiated rice seedlings were then rooted and transferred to rooting medium and cultured at 26°C under light. After approximately 3-4 weeks, when the seedlings reached the rim of the rooting medium tubes, the sealing film was opened, and an appropriate amount of sterile water was injected for hardening. After approximately 3-5 days, these seedlings were identified as transgenic seedlings expressing GUS driven by the Os01g0855000 promoter (SEQ ID NO:4), and were ready for subsequent planting and transgenic identification.

[0131] Example 3: Investigating the expression distribution of gene Os01g0855000 in wild-type rice

[0132] Taking the expression of the Os01g0855000 gene in the aboveground parts and roots of rice as an example, the following steps are included.

[0133] 1. Samples were taken from transgenic seedlings whose GUS (β-glucuronidase gene) expression was driven by the constructed Os01g0855000 promoter (SEQ ID NO:4). A 2mm section was cut from the root tip at a distance of 3.5cm, embedded in 0.5% agarose, trimmed, and sectioned to a thickness of 50μm using a microtome. Similarly, the anthers of the transgenic plants were embedded and sectioned.

[0134] 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) using DFM, and dilute with GUS buffer to a final concentration of 1 mM working solution. The GUS buffer formula is as follows:

[0135]

[0136] 3. Place the sections in GUS staining solution, evacuate the vacuum pump for 30 minutes, and continue staining at 37°C 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 This indicates that the Os01g0855000 gene is expressed in the root endodermis and leaf stomata of rice ZH11.

[0137] Example 4: Investigating the effect of knocking out the Os01g0855000 gene on rice growth

[0138] 1. Soak the mutant gpat6 seeds and wild-type ZH11 seeds in tap water and place them in a 37℃ oven for germination for two days. Then place them in a 96-well plate with the bottom removed and culture them in tap water.

[0139] 2. After culturing in tap water for three days, transfer to a nutrient solution for one week. The nutrient solution formula is as follows:

[0140] reagents and solutions

[0141] The 1000X Yoshida mother liquors are as follows:

[0142]

[0143]

[0144] 3) Rice seedlings that have grown for 14 days are transplanted into paddy soil and cultivated and observed in a greenhouse. The results are shown in [the table below]. Figure 2 . Figure 2 This indicates that the mutant gpat6, resulting from the knockout of the Os01g0855000 gene, exhibits dwarfism.

[0145] Example 5: Leaf observation of mutant gpat6 and wild-type ZH11

[0146] 1. Take the second leaf of 14-day-old mutant gpat6 and wild-type ZH11 rice seedlings, and take the leaf 10cm from the leaf tip.

[0147] 2. Completely immerse the collected leaves in 10% toluidine blue staining solution and stain at room temperature for 45 minutes. Observe the staining condition, and the results are shown in the table below. Figure 3. Figure 3 The results showed that the Os01g0855000 gene knockout mutant gpat6 plants exhibited leaf cuticle defects, resulting in enhanced penetration of toluidine blue.

[0148] Example 6: Comparison of salt stress tolerance between mutant gpat6 and wild-type ZH11

[0149] 1. Soak the mutant seeds and 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.

[0150] 2. After culturing in tap water for three days, transfer it to a nutrient solution and culture for 10 days. Once it reaches the three-leaf stage, perform salt treatment by adding 150mM NaCl to the nutrient solution. After three days of treatment, take photos and record the results. See below. Figure 4 . Figure 4 This indicates that the mutant gpat6 plant after the Os01g0855000 gene was knocked out exhibits a salt-sensitive phenotype, and its growth is affected by salt stress, resulting in plant cessation.

[0151] 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 glycerol acyltransferase 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 monocotyledonous plant, preferably 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 GPAT2O6 expressing the glycerol acyltransferase (Os01g0855000) is SEQ ID NO:2, which consists of a 5'UTR region, a 3'UTR region, introns, and exons (coding region, i.e., CDS); or The nucleotide sequence of the gene GPAT2O6 expressing the glycerol acyltransferase (Os01g0855000) is SEQ ID NO:3, which is the coding region, i.e., the CDS sequence, in 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 glycerol acyltransferase (Os01g0855000) or its expression gene GPAT2O6 in plants.

6. The application as described in claim 5, characterized in that, The overexpression of glycerol acyltransferase (Os01g0855000) or its expression gene GPAT2O6 is achieved through the following method: A. Cloning the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3 as described in claim 3 into a plasmid vector suitable for expression in Agrobacterium to form a recombinant plasmid, namely the GPAT2O6 overexpression vector, and transforming plants using Agrobacterium-mediated transformation to obtain transgenic plants overexpressing glycerol acyltransferase (Os01g0855000); or B. By using gene editing technology, the gene GPAT2O6 with the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:3 as described in claim 3 is cloned into a plant chromosome to obtain a transgenic plant overexpressing glycerol acyltransferase (Os01g0855000). C. Place the plant gene Os01g0855000 under the regulation of a promoter with the nucleotide sequence SEQ ID NO:4 or a promoter with enhanced function.

7. A promoter for increasing the expression level of the glycerol acyltransferase (Os01g0855000) as described in claim 1, characterized in that, It is selected from: (1) A polynucleotide with the nucleotide sequence SEQ ID NO:4; (2) A polynucleotide whose nucleotide sequence is ≥95% identical to the nucleotide sequence shown in SEQ ID NO:4, 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 glycerol acyltransferase (Os01g0855000) gene expression cassette, characterized in that, It includes the promoter as described in claim 7 and the Os01g0855000 gene located downstream therefrom. A recombinant plasmid, characterized in that it contains the above-mentioned glycerol acyltransferase (Os01g0855000) 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 Os01g0855000, 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 GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when 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 salt-tolerant rice varieties. When the rice genome does not contain the gene GPAT2O6 with the nucleotide sequence SEQ ID NO:2 or SEQ ID NO:3, or when the protein expressed by rice cells does not contain a 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 GPAT2O6 gene: Forward primer RT-PCR-GPAT2O6-F: CGTTCGTCTACGTGACCTACAT (SEQ ID NO:5), Reverse primer RT-PCR-GPAT2O6-R: GAGGTAGGCCTCCTTGCATA (SEQ ID NO:6).

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