CN116042649B

CN116042649B - Non-secretory small molecule peptide encoding cysteine-rich small molecule peptide, encoding gene and application thereof

Info

Publication number: CN116042649B
Application number: CN202211409247.XA
Authority: CN
Inventors: 蔡肖; 张建宏; 王海涛; 唐丽媛; 李兴河; 张素君; 刘存敬
Original assignee: Institute Of Cotton Hebei Academy Of Agriculture And Forestry Sciences Hebei Special Economic Crop Research Institute Academy Of Agriculture And Forestry Sciences
Current assignee: Institute Of Cotton Hebei Academy Of Agriculture And Forestry Sciences Hebei Special Economic Crop Research Institute Academy Of Agriculture And Forestry Sciences
Priority date: 2022-11-11
Filing date: 2022-11-11
Publication date: 2023-07-21
Anticipated expiration: 2042-11-11
Also published as: CN116042649A

Abstract

The invention relates to the technical field of plant genetic engineering, and provides a non-secretory small molecular peptide rich in cysteine, a coding gene and application thereof, wherein the coding gene is named GhTYSTM 6A, the nucleotide sequence of the gene is shown as SEQ ID NO.1, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 2. The GhTYS 6A gene is over-expressed in Arabidopsis thaliana, and the resistance of transgenic Arabidopsis thaliana to verticillium wilt is obviously improved. The GhTYSTM 6A is silenced in Ji cotton 2016, a disease-resistant cotton variety, and the resistance of cotton plants to verticillium wilt is obviously reduced. Therefore, the GhTYS 6A gene is favorable for improving the verticillium wilt resistance of plants, and provides a theoretical basis for cultivating verticillium wilt resistant plant varieties.

Description

Non-secretory small molecule peptide encoding cysteine-rich small molecule peptide, encoding gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to a non-secretory small-molecule peptide which codes for cysteine-rich and a coding gene and application thereof.

Background

The verticillium wilt of cotton belongs to a typical soil-borne vascular bundle systematic disease, is the most serious disease in cotton production in China, and affects sustainable development of cotton production in China. The cotton verticillium wilt field control difficulty is high, and the cultivation of disease-resistant varieties is the most economical and effective measure. Although some disease-resistant cotton varieties are cultivated through the efforts of scientific researchers, the disease resistance of the cultivated disease-resistant varieties is gradually lost after the popularization and the planting of the cultivated disease-resistant varieties for a plurality of years due to the rapid mutation of verticillium wilt bacteria. In addition, upland cotton lacks of high verticillium wilt resistant germplasm resources, the traditional breeding period is long, the efficiency is low, and the cotton verticillium wilt resistant breeding in China is slow in progress.

The transgenic technology is used as a rapid and effective method for cotton genetic improvement, and brings great development space for cotton genetic breeding. The method has the advantages of deep excavation, research on the verticillium wilt resistance genes of cotton, understanding of the molecular mechanism of verticillium wilt resistance of cotton, and providing theoretical basis and target genes for improving verticillium wilt resistance of cotton by using a biological breeding method.

Small molecule polypeptides are widely found in eukaryotic organisms and are involved in various aspects of plant growth and development, defense and stress response. Depending on whether secretion is divided into two categories: a secreted polypeptide, a non-secreted polypeptide. The CYSTM (Cysteine-rich Transmembrane Module ) is a family of small molecule proteins, which belongs to a novel non-secreted Cysteine-rich peptide, and The functions and action mechanisms of most members are still unclear.

Disclosure of Invention

The invention provides a non-secretory small molecular peptide which codes for cysteine-rich and a coding gene and application thereof, which are beneficial to solving the problems of high field control difficulty of cotton verticillium wilt and slow breeding progress of cotton verticillium wilt resistance.

The technical scheme of the invention is as follows:

a cysteine-rich non-secreted small molecule peptide encoding gene having a nucleotide sequence of 1) or 2):

1) A nucleotide sequence shown as SEQ ID NO. 1;

2) The nucleotide sequence shown as SEQ ID NO.1 has the same function obtained by replacing, deleting or inserting one or more nucleotides.

A cysteine-rich non-secreted small molecule peptide encoded by said gene having an amino acid sequence as set forth in 1) or 2):

1) An amino acid sequence shown as SEQ ID NO.2,

2) The amino acid sequence shown as SEQ ID NO.2 has the same function obtained by replacing, deleting or inserting one or more amino acids.

The application of the coding gene of the cysteine-rich non-secretory small molecule peptide in preventing and treating plant verticillium wilt.

The application of the non-secreted small molecule peptide rich in cysteine in preventing and treating plant verticillium wilt.

As a further technical scheme, the gene is over-expressed in the plant body, so that the verticillium resistance of the plant is improved.

The beneficial effects of the invention are as follows:

1. the invention clones a small molecule peptide gene encoding non-secretion type cysteine-rich from the cDNA of the leaf of Ji-cotton 2016 of a upland cotton variety for the first time, and the gene is named as GhTYS 6A by comparing with a upland cotton reference genome and a homologous sequence in Arabidopsis thaliana, so that the expression of the gene is closely related to the infection of verticillium wilt, and a theoretical basis is provided for cultivating a verticillium wilt-resistant plant variety.

2. The GhTYS 6A gene is over-expressed in the Arabidopsis, and the verticillium resistance of the transgenic Arabidopsis is obviously improved. The GhTYSTM 6A is silenced in Ji cotton 2016, a disease-resistant cotton variety, and the resistance of cotton plants to verticillium wilt is obviously reduced.

3. The GhTYS 6A gene and the encoding protein thereof can be applied to preventing and treating cotton verticillium wilt and improving the verticillium wilt resistance of plants, can be applied to the aspect of verticillium wilt resistance genetic improvement or molecular breeding of cotton and other plants, and have important significance for plant verticillium wilt resistance genetic improvement.

Drawings

The invention will be described in further detail with reference to the drawings and the detailed description.

FIG. 1 is a phylogenetic tree analysis of GhTYS 6A and different proteins in example 1;

FIG. 2 is a graph of GhTYS 6A subcellular localization in example 1;

FIG. 3 is a graph showing the overexpression of the 35S promoter-driven GhTYS 6A in transgenic Arabidopsis in example 2;

FIG. 4 is a graph showing the identification of verticillium resistance of an Arabidopsis thaliana infected with Verticillium dahliae in example 2;

FIG. 5 is a graph of verticillium dahliae resistance identification of a VIGS-silenced cotton plant infected with Verticillium dahliae of example 3.

Detailed Description

The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1: cloning, sequencing, evolutionary analysis and subcellular localization of the GhTYS 6A Gene

(1) Plant material

Ji cotton 2016

(2) Experimental method

Extracting Ji cotton 2016 leaf RNA by Trizol method, and referring to cDNA synthesis reverse transcription kit PrimeScript ^TM The RT reagent Kit (TaKaRa) instructions reverse transcribe RNA into cDNA.

(3) Sequence analysis

CDS sequence of GhTYS 6A gene is cloned in cDNA of Ji cotton 2016 leaf, full length 237bp, nucleotide sequence is shown in SEQ ID NO. 1:

ATGAATCACAACCAGCAACACTTCAATCAATCATCAGCAGCTGCATACCCACCACCACCACCCTCCACGGCGCCAGGACCCTACGTGACTGCACCGCCGGCCGGTTACCCGATGACCAAAGACGAGTACAGTCAGCAAAATCCGGCTGCTGTCGAAACCAAGTCCAGAGGTGATGGATTCTGGAAAGGATGTTGTGCTGCCCTTTGCTGTTGTTGCCTTCTGGATGCATGCTTTTGA

the coded protein comprises 78 amino acids, and the amino acid sequence is shown in SEQ ID NO. 2:

MNHNQQHFNQSSAAAYPPPPPSTAPGPYVTAPPAGYPMTKDEYSQQNPAAVETKSRGDGFWKGCCAALCCCCLLDACF

the amino acid sequence was analyzed in the SMART and Pfam protein databases and the GhTYS 6A protein was found to contain CYTSTM domain (PF 12734) belonging to the CYSTM family of members. The GhTYS 6A gene was found to be located on the A07 chromosome by comparison of upland cotton genomes using localized blast-2.12.0 software. The NCBI performs on-line blastp comparison, and finds that the GhTYS 6A protein has the highest homology with XP_ 016703581.1. XP_016703581.1 is also derived from upland cotton (Gossypium hirsutum), the protein function is not annotated, and the function of homologous genes thereof is not reported in other species at present.

(4) GhTYSTM 6A evolution analysis

Taking the NCBI to obtain the online blastp comparison result, wherein the homology is more than 75% and the value of Evalue is less than 1e ^-5 The proteins were grouped into 3 classes-Group I, group II, group III (as shown in FIG. 1), ghTYS 6A was grouped into one class (Group I) with other proteins from the genus cotton of the family Malvaceae, and they were most recently related to Hibiscus of the family Malvaceae.

(5) Subcellular localization

In order to study subcellular localization of the GhTYTM 6A gene, primers GhTYTM 6A-GFP-F and GhTYTM 6A-GFP-R were used to amplify the cDNA of Ji cotton 2016 leaf, the PCR amplified product was digested with restriction enzymes EcoRI and BamHI, and the plasmid vector pCAMBIA2300-35S-GFP was used to link the PCR recovered product with the vector backbone, and the CDS sequence was constructed into pCAMBIA2300-35S-GFP expression vector to obtain the 35S: ghTYTM 6A-GFP fusion expression vector.

GhCYSTM6A-GFP-F：5’-TTGAATTCATGAATCACAACCAGCAACACTTC-3', wherein underlined GAATTC indicates the recognition sequence of the restriction enzyme EcoRI, TT being a protecting base.

GhCYSTM6A-GFP-R：5’-TTGGATCCTCAAAAGCATGCATCCAGAACG-3', wherein underlined GGATCC indicates the recognition sequence of the restriction enzyme BamHI, TT is a protecting base.

By utilizing a tobacco leaf transient transformation system, 35S: ghCDSTM 6A-GFP protein fusion expression vector and 35S: GFP empty load are respectively injected into Nicotiana benthamiana leaves for transient transformation, subcellular localization analysis is carried out after 2-3 days according to the luminous condition of the fusion protein observed under a laser confocal microscope, and the result shows that the GhCDSTM 6A protein has localization signals in cell nuclei and cell membranes, as shown in figure 2.

Example 2: identification of verticillium dahliae resistance of transgenic arabidopsis thaliana under stress

The primers GhTYTM 6A-OE-F and GhTYTM 6A-OE-R are used for carrying out PCR amplification on Ji cotton 2016 leaf cDNA, a restriction enzyme XhoI and XbaI double-restriction enzyme plasmid vector pART-CAM is adopted for carrying out recombination connection on the PCR recovered product and a vector skeleton, and a GhTYTM 6A gene overexpression vector pART-CAM-GhTYTM 6A is constructed.

GhCYSTM6A-OE-F：5’-GGAGAGGACACGCTCGAGATGAATCACAACCAGCAACACTTC-3', wherein the underlined CTCGAG represents the recognition sequence of the restriction enzyme XhoI, GGAGAGGACACG is a homologous sequence.

GhCYSTM6A-OE-R：5’-TTAAAGCAGGACTCTAGATCAAAAGCATGCATCCAGAACG-3', wherein the underlined TCTAGA indicates the recognition sequence for the restriction enzyme XbaI, TTAAAGCAGGAC is a homologous sequence.

Transforming GhTYTM 6A gene over-expression vector into Arabidopsis thaliana by dipping flower method, and harvesting after seed maturation to obtain T ₀ Sterilizing the seeds, placing the seeds in an MS culture medium, vernalizing the seeds for 2 days at 4 ℃, then transferring the seeds into an incubator with light for 16h and darkness for 8h at 25 ℃, transplanting the seeds into nutrient soil after the seeds grow for 7 days, and performing PCR detection on positive plants after 21 days of growth of arabidopsis thaliana, namely the T-shaped plants ₁ Seed, and so on, through generation and purification to T3, 3 GhTYS 6A oversurface are obtained through semi-quantitative detectionTransgenic homozygous lines: OE5, OE10 and OE14, as shown in fig. 3.

In order to identify verticillium wilt resistance of transgenic arabidopsis lines, arabidopsis grown for 7d on an MS culture medium is transferred into nutrient soil and cultured for 3 weeks under normal growth conditions, wild Type (WT) with orderly and consistent growth vigor is selected, verticillium dahliae pathogenic bacteria, namely verticillium dahliae V991, are inoculated to the transgenic arabidopsis, plant morbidity and phenotype change are observed after 20 days, and verticillium wilt fingers are counted.

The disease-level grading criteria were:

level 0: no morbidity exists;

stage 1: less than or equal to 25 percent of leaf disease;

2 stages: 26% -50% of leaf disease;

3 stages: 51% -75% of leaf disease;

4 stages: and more than or equal to 75 percent of the leaves are ill.

Disease refers to a calculation formula:

SIGMA (number of individual disease stages. Times. Grade of corresponding disease stage)/total number of investigation. Times. Highest disease stage ]. Times.100.

The results showed that after 20 days of inoculation with verticillium dahliae V991, the verticillium wilt symptoms such as verticillium wilt, wilting, necrosis, etc. of the gcystm 6A transgenic arabidopsis leaves were lighter than those of the wild type (fig. 4), the left side of fig. 4 being the wild type and the right side being the transgenic group.

TABLE 1 verticillium wilt of transgenic Arabidopsis thaliana and wild Arabidopsis thaliana

Plants and methods of making the same	Disease refers to (%)
		Wild Arabidopsis thaliana	31.67±6.61
Transgenic Arabidopsis thaliana	55.28±6.79*

In the table, P <0.5

The transgenic Arabidopsis verticillium finger (31.67% + -6.61) was significantly lower than the wild type verticillium finger (55.28% + -6.79) (P < 0.5), with significant differences. The results indicate that the transgenic Arabidopsis thaliana of GhTYS 6A has higher verticillium wilt resistance than the wild type, and the overexpression of GhTYS 6A is related to the improvement of the disease resistance of plants.

Example 3: identification of verticillium dahliae resistance of cotton plants with VIGS-silenced GhTYSTM 6A gene under stress of verticillium dahliae

The disease-resistant Ji cotton 2016 is used as a material and contains an empty vector pTRV2: 00. the gene silencing vector pTRV2 of interest: the agrobacteria liquid of GhCYTM6A is respectively mixed with the agrobacteria liquid of the auxiliary carrier pTRV1 in equal proportion, and then the mixed bacteria liquid is respectively inoculated to the back of the cotyledon which is just flattened of the cotton seedling. The inoculated cotton plants are placed in an incubator at 25 ℃ for dark culture for 24 hours, and then are changed into culture conditions of 16h illumination and 8h darkness. After 10 days of inoculation, the silencing plants and the control empty carrier plants are respectively inoculated with verticillium dahliae V991 by adopting a root irrigation method, the phenotypic change of the plants after being inoculated with verticillium for 2 weeks is observed, and verticillium wilt fingers are investigated.

The disease-level grading criteria were:

level 0: no symptoms;

stage 1: 1 leaf of true leaf;

2 stages: 2 pieces of true leaves are ill;

3 stages: 3 true leaves are ill;

4 stages: 4 or more true leaves are ill.

Disease refers to a calculation formula: the total number of each stage of disease was examined × the highest stage of disease (4) ] × 100.

TABLE 2 verticillium wilt of transgenic Arabidopsis thaliana and wild Arabidopsis thaliana

Plants and methods of making the same	Disease refers to (%)
		No-load plant	63.33±3.36
Gene silencing plants	76.67±2.89

In the table, P <0.5

The results showed that the verticillium finger (76.67% ± 2.89) of the ghcytm6A gene-silenced plants was significantly higher than the verticillium finger (63.33% ± 3.36) of the empty vector-inoculated plants (P < 0.5) 14 days after V991 inoculation. From the phenotype, most leaves of the silent plants turn yellow, some plant leaves are all yellow, necrotic and shed, and almost only stems remain, while the control plants inoculated with the empty vector mainly show the yellowing, shrinkage necrosis and even shedding of the lower leaves, and new leaves can still be basically kept normal (figure 5), which indicates that the GhTYTM 6A silencing can reduce the resistance of cotton plants to verticillium wilt.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims

1. The application of the coding gene of the non-secretory small molecule peptide rich in cysteine in preventing and treating plant verticillium wilt is characterized in that the nucleotide sequence of the coding gene is shown as SEQ ID NO. 1.

2. The application of a non-secretory small molecule peptide rich in cysteine in preventing and treating plant verticillium wilt is characterized in that the small molecule peptide is obtained by encoding a gene as set forth in claim 1, and the amino acid sequence of the small molecule peptide is shown as SEQ ID NO. 2.

3. The use according to claim 1 or 2, wherein the gene is overexpressed in plants to increase verticillium resistance in plants.