Salt stress negative regulatory factor gmzat8 derived from soybean and application thereof

Abstract

The application relates to the technical field of plant genetic engineering and molecular breeding, and particularly discloses a salt stress negative regulation factor GmZAT8 derived from soybeans, characterized in that the nucleotide sequence of the negative regulation factor GmZAT8 is shown as SEQ ID NO:1, and the coding protein is shown as SEQ ID NO:2. GmZAT8 The application first discloses the salt stress negative regulation function of a C2H2 zinc finger protein ZAT subfamily member gene of soybeans, enriches the genetic resources of a plant salt stress negative regulation network, and provides new research materials and theoretical foundations for in-depth analysis of the salt stress response mechanism of soybeans and other crops. GmZAT8 Based on the function of negatively regulating salt tolerance, by knocking out or inhibiting the expression of the gene through CRISPR / Cas9 gene editing technology, a new plant material with significantly improved salt tolerance can be expected.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering and molecular breeding technology, specifically relating to a negative regulator of salt stress, GmZAT8, derived from soybean, and its application. Background Technology

[0002] Soil salinization is one of the major abiotic stressors limiting global agricultural production. High-salt environments inhibit plant growth and development through osmotic stress, ion toxicity, and oxidative damage, leading to reduced crop yields and even death. Breeding salt-tolerant crop varieties is an effective strategy to address the threat of salinization, and a deep understanding of the molecular mechanisms of plant salt tolerance is a crucial foundation for molecular breeding.

[0003] In recent years, researchers have isolated and identified a large number of salt stress response genes from various plants, mainly including: ion transporter genes (such as... SOS1 , NHX1 ), osmotic regulatory substance synthesis enzyme genes (such as P5CS ), and various transcription factor genes (such as DREB , NAC , bZIP , WRKY wait).

[0004] Negative regulatory factors reveal the intricate regulatory network by which plants actively inhibit growth or initiate senescence under salt stress, thus completing the overall picture of stress response. Negative regulatory genes are ideal targets for gene editing—knocking out or downregulating these genes using technologies such as CRISPR / Cas9 can directionally improve crop salt tolerance, often without introducing exogenous fragments and making it easier to pass safety assessments. Therefore, cloning and validating salt stress negative regulatory factors from crops such as soybean, and clarifying their functions, is of great significance for elucidating salt tolerance mechanisms and creating salt-tolerant germplasm resources.

[0005] There are few reported genes that negatively regulate salt stress in soybean, and whether members of the ZAT subfamily of the C2H2 zinc finger protein family participate in the negative regulation of salt stress in soybean remains a blank. The lack of diverse negative regulatory gene resources limits our understanding of the comprehensive mechanisms of the salt tolerance negative regulatory network. While existing methods have verified the phenotype of "overexpression leading to salt sensitivity," their technical endpoints typically remain at the level of gene function identification, i.e., "discovering a negative regulatory gene." There has been no further development of this salt sensitivity phenotype into a tool or model with practical application value. Existing methods usually only vaguely mention that "salt tolerance can be improved by knocking out or inhibiting this gene," but lack operable technical solutions for specific application scenarios (such as gene editing target design, and construction of salt-sensitive indicator plants). Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art, overcoming the deficiencies of the lack of soybean ZAT subfamily salt stress negative regulatory gene resources and the lack of functional application tools based on salt-sensitive phenotypes, and to provide a soybean-derived salt stress negative regulatory factor GmZAT8 and its applications, including its various uses in constructing salt-sensitive reporter plants and as a gene editing target. We used soybean heterologous expression in Arabidopsis thaliana to achieve this. GmZAT8 The gene was extracted, and homozygous overexpression lines with significantly increased expression levels were obtained. A search revealed no results regarding... GmZAT8 Any reports of genes involved in the negative regulation of salt stress.

[0007] The purpose of this invention is to provide a soybean C2H2 zinc finger protein family gene. GmZAT8 Validating its negative regulatory function under salt stress, and establishing one or more of the following applications: constructing transgenic plants with enhanced salt sensitivity (such as Arabidopsis thaliana) as a standardized salt stress sensitivity model, which can be used as a universal salt stress sensitivity control material in the laboratory; providing gene editing targets for salt tolerance improvement: based on the negative regulatory function, GmZAT8 As target genes, new plant germplasm with improved salt tolerance can be obtained through gene knockout or expression repression techniques; enriching the theory of negative regulation of plant salt stress: providing new gene resources and experimental evidence for elucidating the regulatory mechanism of C2H2 zinc finger protein in plant stress response.

[0008] The present invention provides the following technical solution: a negative regulator of salt stress, GmZAT8, derived from soybean, wherein the nucleotide sequence of the negative regulator GmZAT8 is shown in SEQ ID NO:1, and the encoded protein is shown in SEQ ID NO:2.

[0009] The above-mentioned negative regulator of salt stress, GmZAT8, derived from soybean, is applied to salt stress-sensitive control materials commonly used in breeding laboratories.

[0010] A method for cultivating salt stress-sensitive control materials for laboratory use, characterized by overexpression of the gene GmZAT8 in plants.

[0011] Furthermore, the above method includes the following steps: (1) Construction of overexpression vector The CDS of GmZAT8 was cloned into a plant overexpression vector using Gateway technology. First, the CDS was inserted into the entry vector, then recombined via LR reaction into the target vector pEarleyGate 103, which contains a 35S promoter and reporter tag, to obtain the final expression vector. 35S::GmZAT8 .

[0012] (2) Obtaining transgenic Arabidopsis The above expression vector was transformed into plants using the Agrobacterium inflorescence inoculation method; T0 generation transgenic plants were obtained through resistance screening, and homozygous T3 generation transgenic lines were obtained through continuous self-pollination screening.

[0013] A recombinant expression vector comprising the nucleotides of claim 1.

[0014] The above-mentioned application of GmZAT8, a negative regulator of salt stress derived from soybean, in improving plant salt tolerance.

[0015] The above applications are achieved by knocking out or inhibiting the expression of the GmZAT8 gene.

[0016] Compared with the prior art, the beneficial effects of the present invention are: 1. The GmZAT8 involved in this invention belongs to the ZAT subfamily of C2H2 zinc finger proteins, and the function of this family in the negative regulation of salt stress in soybeans has not been previously reported. This invention fills this gap, enriches the genetic basis of the salt tolerance negative regulatory network, and provides a novel gene resource for subsequent mechanism research and breeding applications.

[0017] 2. This invention provides new experimental evidence and gene resources for a deeper understanding of the negative regulatory network of plant salt stress response, and helps to improve the molecular mechanisms of plant stress adaptation.

[0018] 3. The GmZAT8 overexpressing Arabidopsis strain obtained in this invention can be used as a standardized salt-sensitive control material for: negative controls in salt stress experiments within or between laboratories; and sensitive background material for other researchers to verify the function of salt-tolerant genes.

[0019] 4. Based on the negative regulatory function of GmZAT8, this invention provides a candidate target for obtaining plants with improved salt tolerance by knocking out or inhibiting this gene through gene editing technology. Attached Figure Description

[0020] Figure 1 The image shown is from Example 2. GmZAT8 Identification of expression levels in transgenic lines overexpressing Arabidopsis thaliana; Figure 2 The image shown is from Example 3. GmZAT8 Verification of salt sensitivity phenotype in Arabidopsis thaliana overexpression. A represents the growth status of three lines: wild-type and independent overexpression, under clean water conditions. B represents the phenotypic status of the wild-type and the independent overexpression lines under salt-treated clean water conditions; Figure 3 The figure shows the survival rate statistics of wild-type and overexpression lines after salt treatment in Example 3. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0022] Example 1: Example 1 GmZAT8 Construction of Arabidopsis thaliana overexpression Using soybean (Glycine max) Williams 82 seedling cDNA as a template, the full-length coding sequence of the GmZAT8 gene (SEQ ID NO:1) was amplified by PCR using primers (SEQ ID NO:3-4) and confirmed by sequencing. The CDS of GmZAT8 was cloned into a plant overexpression vector using Gateway technology. The recovered product was inserted into the pENTR / D-TOPO vector via TOPO cloning and sequenced for verification. GmZAT8 was then transferred into the plant expression vector pEarleyGate 103 (containing a 35S promoter) via LR recombination to obtain the recombinant plasmid. 35S::GmZAT8.

[0023] SEQ ID NO:3ATGAAGAGAGGCAGAGAAGAG SEQ ID NO:4AATGAAACAATTGAGGACGGGC Example 2 GmZAT8 Obtaining Arabidopsis thaliana overexpression materials The above-mentioned vector was transformed into Arabidopsis wild-type Col-0 using the Agrobacterium inflorescence inoculation method. T0 generation plants were obtained through glufosinate resistance screening, and homozygous transgenic lines were obtained through continuous self-pollination to the T3 generation. Confirmation was performed by qRT-PCR. GmZAT8 High gene expression was observed, and the lines with the highest expression levels (OE-2, 3, 4) were selected for subsequent experiments. Figure 1 ).

[0024] Example 3 GmZAT8 Validation of salt sensitivity phenotype by overexpression of Arabidopsis thaliana Five-day-old wild-type and OE-1 seedlings were transplanted into soil and cultured under long-day conditions (16 h light / 8 h darkness) for two weeks. They were then treated with 200 mM NaCl for 13-17 days, followed by a return to watering for 3-5 days after treatment. Observations were made when significant differences were observed (at least 12 plants per group, with three replicates). Survival rates were recorded. Results showed that the overexpression lines exhibited severe leaf wilting and yellowing. Figure 2The survival rates (approximately 44.44%, 56%, and 44.44% for the three overexpression lines, respectively) were significantly lower than those of the wild type (approximately 72%). Figure 3 ).show GmZAT8 Overexpression significantly enhanced the salt sensitivity of Arabidopsis thaliana.

Claims

1. A negative regulator of salt stress, GmZAT8, derived from soybean, characterized in that... The nucleotide sequence of the negative regulatory factor GmZAT8 is shown in SEQ ID NO:1, and the encoded protein is shown in SEQ ID NO:

2.

2. The application of GmZAT8, a negative regulator of salt stress derived from soybean as described in claim 1, in salt stress-sensitive control materials commonly used in breeding laboratories.

3. A method for cultivating a laboratory-standard salt stress-sensitive control material, characterized in that, The gene GmZAT8 is overexpressed in plants.

4. The method according to claim 3, characterized in that, It includes the following steps: (1) Construction of overexpression vector The CDS of GmZAT8 was cloned into a plant overexpression vector using Gateway technology. First, the CDS was inserted into the entry vector, then recombined via LR reaction into the target vector pEarleyGate 103, which contains a 35S promoter and reporter tag, to obtain the final expression vector. 35S::GmZAT8; (2) Obtaining transgenic Arabidopsis The above expression vector was transformed into plants using the Agrobacterium inflorescence inoculation method; T0 generation transgenic plants were obtained through resistance screening, and homozygous T3 generation transgenic lines were obtained through continuous self-pollination screening.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleotides described in claim 1.

6. The application of GmZAT8, a negative regulator of salt stress derived from soybean, as described in claim 1, in improving plant salt tolerance.

7. The application according to claim 6, characterized in that: This is achieved by knocking out or suppressing the expression of the GmZAT8 gene.

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