阅读说明：本技术 植物抗盐蛋白MsVNI1及编码基因和应用 (Plant salt-resistant protein MsVNI1, and coding gene and application thereof ) 是由 付春祥 杨瑞娟 刘文文 张晓伟 曹英萍 白史且 周传恩 吴振映 王亚梅 姜珊珊 于 2020-05-18 设计创作，主要内容包括：本发明涉及一种植物抗盐蛋白MsVNI1及编码基因和应用,属于植物基因工程技术领域,植物抗盐蛋白MsVNI1氨基酸序列SEQ ID NO.2所示,核苷酸序列如SEQ ID NO.1所示。本发明对植物抗盐蛋白MsVNI1进行分子调控,能够显著增加拟南芥的耐盐品质,提高紫花苜蓿叶片大小。对于牧草及其他作物生物量和抗逆性的遗传改良具有重要的参考意义。(The invention relates to a plant salt-resistant protein MsVNI1, a coding gene and application, and belongs to the technical field of plant genetic engineering, wherein the amino acid sequence of the plant salt-resistant protein MsVNI1 is shown as SEQ ID No.2, and the nucleotide sequence is shown as SEQ ID No. 1. According to the invention, the plant salt-resistant protein MsVNI1 is subjected to molecular regulation, so that the salt-resistant quality of arabidopsis can be remarkably improved, and the size of alfalfa leaves can be improved. Has important reference significance for the genetic improvement of the biomass and the stress resistance of pasture and other crops.)

1. A plant salt-resistant protein MsVNI1 is characterized in that the amino acid sequence is shown in SEQ ID NO. 2.

2. The gene for coding the plant salt-resistant protein MsVNI1 as claimed in claim 1, wherein the nucleotide sequence is shown as SEQ ID No. 1.

3. A recombinant vector pCABIA1300-MsVNI1-cGFP comprising the plant salt-resistant protein MsVNI1 gene according to claim 2.

4. The use of the recombinant vector of claim 3 for controlling plant salt tolerance and leaf size.

5. A method for improving the expression level of a plant salt-resistant protein MsVNI1 in a plant body, wherein an overexpression vector is constructed by using the MsVNI1 gene full-length sequence of claim 1, and the expression level of the MsVNI1 in arabidopsis and alfalfa bodies is improved.

6. The application of the plant salt-resistant protein MsVNI1 in the aspects of regulating and controlling the salt tolerance and the leaf size of a plant as claimed in claim 1, wherein the method comprises the steps of obtaining a CDS region sequence of a plant salt-resistant protein MsVNI1 gene, designing amplification primers MsVNI1-F and MsVNI1-R at two sides of the plant salt-resistant protein MsVNI1 according to a clover variety Tribulus alfalfa of which the genome is sequenced, sequencing the amplified fragments to obtain a full-length sequence 1074bp of the plant salt-resistant protein MsVNI 1; the obtained full-length sequence fragment is recombined and integrated into an expression vector pEG100 based on Gateway technology; transferring the agrobacterium tumefaciens EHA105 mediated genetic transformation method into plants, and screening the resistance of glufosinate ammonium to obtain resistant regeneration plants.

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a plant salt-resistant protein MsVNI1, a coding gene and application.

Background

Alfalfa (Medicago sativa L.) is a perennial legume forage, and is the most widespread forage variety in China. Because of high yield, high nutritional quality and strong palatability, the forage grass is known as the king of pasture and plays an important role in the development of animal husbandry in China. In addition, the alfalfa root system is very developed and has strong abiotic stress resistance. With the large-area cultivation and intensive production of alfalfa, the demand for new alfalfa varieties with high quality, high resistance and high yield is urgent. The quality of the alfalfa is improved by utilizing a genetic engineering means, and the alfalfa varieties with high quality, high yield and high stress resistance can be cultured more quickly, so that the development of pasture and animal husbandry is promoted.

The salinization of land is a common problem which puzzles China and even the world, and about 10 hundred million hm is existed all over the world²The salinized land of (1) has the highest salinization degree in China, which accounts for about 1/10 and coastal areas, and has become an important factor influencing agricultural production. Research on plant salt tolerance has gradually become a global focus of attention in recent years. Based on the principle of 'no competition for grains with people and no competition for land with grains', the pasture with strong salt tolerance is applied to the saline-alkali land improvement through genetic improvement, so that the dual aims of saving the cultivated land area and improving the animal husbandry development in the saline-alkali area are fulfilled.

The transcription factor is positioned at the upstream of gene expression regulation, and can realize the common regulation of a plurality of traits, so that the transcription factor becomes one of candidate targets for plant multi-trait regulation. The findings in the Arabidopsis thaliana study

VND-INTERACTING1 and 2(VNI1/2) are NAC transcription factors that interact with VND7(VASCULAR-RELATED NAC-DOMAINPROTEIN 7), and among them, the study of VNI1 has been rarely reported (Yamaguchi et al, VND-INTERACTING2, a NAC domain transcription factor, novel regulation expression in Arabidopsis plant Cell,22: 1249-1263). The NAC protein family is a plant-specific transcription factor superfamily, and plays an important role in biological processes such as plant growth and development, metabolic regulation, stress and the like. Currently, in the research of improving the salt tolerance of plants, most genes cannot simultaneously meet the dual requirements of salt tolerance and plant yield preservation. Therefore, the gene function of the alfalfa MsVNI1 is researched by using a modern biological method, and the possibility is provided for improving the salt tolerance of the alfalfa and ensuring the crop yield.

Disclosure of Invention

Aiming at the technical requirements in the field of plant salt tolerance in the background technology, the invention aims to provide a plant salt-resistant protein MsVNI1, a coding gene and application thereof, and solves the problems that the existing plant salt-resistant gene resource library is insufficient and the requirements of improving the yield and quality of plants and molecular design cannot be met at the same time.

The invention is realized by the following technical scheme:

a plant salt-resistant protein MsVNI1, the amino acid sequence of which is shown in SEQ ID NO. 2.

The invention also provides a gene for coding the plant salt-resistant protein MsVNI1, and the nucleotide sequence of the gene is shown in SEQ ID NO. 1.

The invention also provides a recombinant vector containing the plant salt-resistant protein MsVNI1 gene

pCABIA1300-MsVNI1-cGFP。

The invention also provides application of the recombinant vector in regulating and controlling the salt tolerance of plants and the size of leaves.

The invention also provides a method for improving the expression level of the plant salt-resistant protein MsVNI1 in the plant body, the method utilizes the MsVNI1 gene full-length sequence to construct an over-expression vector, and the expression level of the MsVNI1 in the arabidopsis and alfalfa bodies is improved.

The invention also provides application of the plant salt-resistant protein MsVNI1 in regulation and control of plant salt-resistant performance and leaf size.

The application method comprises the steps of obtaining a CDS region sequence of a plant salt-resistant protein MsVNI1 gene, and designing amplification primers MsVNI1-F and MsVNI1-R at two sides of a plant salt-resistant protein MsVNI1 according to a Medicago sativa variety of which genome sequencing is completed. Sequencing the amplified fragment to obtain a full-length sequence 1074bp of a plant salt-resistant protein MsVNI 1; further, the obtained full-length sequence fragment is recombined and integrated into an expression vector pEG100 based on Gateway technology; transferring the agrobacterium tumefaciens EHA105 mediated genetic transformation method into plants, and screening the resistance of glufosinate ammonium to obtain resistant regeneration plants. Finally determining a positive transgenic plant through PCR analysis; the germination experiment of the arabidopsis seeds shows that the salt tolerance of the transgenic plants is improved; leaves of alfalfa transgenic plants slightly over-expressing horizontal plants become bigger.

The core characteristics and the inventive concept of the invention comprise:

1. the expression level of the plant salt-resistant protein MsVNI1 in the alfalfa body is improved by means of genetic engineering through an overexpression technology, and a remarkable effect can be obtained in a short time;

2. the invention starts from the genes for regulating and controlling the salt tolerance and various characters of the plants, ensures the crop yield, analyzes the regulation and control mechanism of the salt stress of the alfalfa gene, and provides more gene resources for the salt tolerance resources of the plants.

Compared with the prior art, the invention has the following beneficial effects:

1. the obtained plant salt-resistant protein MsVNI1 gene is a key gene for regulating and controlling the salt-resistant quality of alfalfa, and has important significance for obtaining plants with high transformation efficiency by a molecular breeding means;

2. according to the invention, the plant salt-resistant protein MsVNI1 is subjected to molecular regulation, so that the salt-resistant quality of arabidopsis thaliana can be remarkably improved, and the size of alfalfa leaves can be improved. The method has important referential significance for the genetic improvement of the biomass and the stress resistance of pasture and other crops;

3. the genetically improved plant produced by the invention can be integrated into a conventional breeding project, thereby providing a new germplasm resource for variety cultivation of pasture crops.

Drawings

FIG. 1 shows the electrophoretogram of the plant salt-resistant protein MsVNI1 clone in alfalfa.

FIG. 2 is a sequence alignment chart of a plant salt-resistant protein MsVNI1 in alfalfa and a Tribulus terrestris homologous gene MtVNI 1.

FIG. 3 is a diagram of the alfalfa pEG100-MsVNI1-OE expression vector.

FIG. 4MsVNI1-OE transgenic Arabidopsis and MsVNI1 gene quantitative PCR results in wild type plants.

FIG. 5 statistical plots of salt stress treated seed germination rates for MsVNI1-OE transgenic Arabidopsis and wild type plants.

FIG. 6MsVNI1-OE transgenic alfalfa and wild type plants MsVNI1 gene quantitative PCR results.

FIG. 7MsVNI1-OE transgenic alfalfa plant phenotype and leaf size phenotype plots.

Detailed Description

The present invention will be described in further detail with reference to the following specific embodiments and the accompanying drawings. Materials, reagents, molecular marker probes, and the like used in the following examples are commercially available from companies unless otherwise specified.