CN113528558A - Application of gene GhSINAs in prevention and treatment of cotton verticillium wilt - Google Patents

Abstract

The invention relates to the technical field of plant disease control, in particular to application of a gene GhSINAs in cotton verticillium wilt control. The invention provides application of genes GhSINAs in preventing and treating cotton verticillium wilt, wherein the genes GhSINAs comprise any one or more of genes GhSINA7, genes GhSINA8 and genes GhSINA 9; the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2; the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4; the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6. In the research, the invention discovers that each over-expression transgenic line of the three genes improves the resistance to verticillium wilt, but the silencing of each gene inhibits the defense capacity to pathogen infection, which indicates that GhSINA7, GhSINA8 and GhSINA9 play a positive regulation role in cotton defense against verticillium wilt.

Description

Application of gene GhSINAs in prevention and treatment of cotton verticillium wilt

Technical Field

The invention relates to the technical field of plant disease control, in particular to application of a gene GhSINAs in cotton verticillium wilt control.

Background

Verticillium dahliae is a very destructive soil-borne fungal pathogen that causes verticillium wilt, which affects the growth and development of over 200 plants, including cotton, an important agricultural commodity. As is known, verticillium dahliae is a very difficult-to-control pathogen which has extremely strong infection pathogenicity and strong survival capability in soil, so that the verticillium dahliae can penetrate through main root xylem vessels and vascular tissues to seriously block the vessels of plants, and finally the plants are late in development and withered. In addition, the host range of this pathogen is wide, and it can survive in soil for many years even without a host. In cotton production, the breeding of disease-resistant varieties is an effective and practical strategy for controlling the threat of verticillium wilt. However, due to the limited resources of the existing verticillium wilt-resistant strains, it is challenging to develop resistant strains using traditional methods. In recent years, the study of candidate genes for cotton resistance based on genetic engineering has become another strategy for alleviating the infestation of verticillium wilt, and among them, GhMYB1, GbSOBIR1, GhCRR1, GbTSA1 and GhWAK7A have been reported.

The Ubiquitination Protease System (UPS) plays an important role in plant growth and development and defense response of physiological and environmental stresses. The ubiquitination process is usually carried out by the sequential action of three enzymes, ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3), through a 26S proteasome degradation system. And (3) connecting the ubiquitin with a candidate protein substrate to realize the degradation of the target protein. Ubiquitin E1 and E2 are relatively conserved, but E3 is very diverse, as E3 can recruit specific target proteins for ubiquitination. Based on subunit groups, E3 ubiquitin ligase is divided into single and multiple subunits. HECT and RING/U-box type E3 ligases function as single subunits, whereas the Skp-Cullin-F-box complex and the promotion complex generated later in cell division bind substrates by separating subunits with multi-subunit ubiquitin ligases. Due to the different composition of these subunits, the E3 ligase determines the specificity of the ubiquitination candidate substrate.

The SINA protein is a RING-type E3 ligase that contains a RING domain located at the N-terminus, followed by a conserved SINA domain involved in substrate recognition and polymerization. The first identified SINA E3 ligase regulated the differentiation of Drosophila melanogaster photoreceptors. A series of subsequent studies have shown that SINA homologues play a number of extremely important roles in animals, such as tumour suppression, apoptosis, leukaemias, hypoxia response and autoimmunity. Similarly, SINA E3 ligase is involved in various plant growth and development stages and some environmental stress reactions. In Arabidopsis, the synthesis of xanthophylls is mediated by the interaction of SINAT2 and AtRAP2.2. SINAT1 and SINAT2 proteins interact with the autophagy-related protein ATG6 to regulate the autophagy pathway. In rice, silencing drought-induced SINA gene 1(OsDIS1) can significantly improve the drought resistance of rice. In tomato, overexpression of SlSINA4 gene can activate cell death signals associated with defense responses. In bananas, MaSINA1 negatively regulates the stress response to cold stress by mediating stability of masice 1. In transgenic apples, the sensitivity to ABA is enhanced by over-expressing MdSINA2 gene. Under heat stress conditions, the TaSINA E3 ligase increased wheat biomass and yield. However, the function and use of SINA E3 ligase in connection with resistance to greensickness is not yet known.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In order to research the functional action of the E3 ubiquitin ligase gene in resisting verticillium dahliae, the invention identifies three SINA E3 ligase genes GhSINA7, GhSINA8 and GhSINA9 induced by verticillium wilt infection. Of these three genes, each overexpressing transgenic line has increased resistance to verticillium wilt, whereas silencing of each gene suppresses the ability to defend against pathogen infection. These findings indicate that SINA E3 ligase participates in plant defense against pathogenic fungi, and simultaneously provides effective gene resources for breeding verticillium wilt-resistant cotton varieties through posttranslational modification.

Based on the above, the present invention provides the following technical solutions:

the application of gene GhSINAs in preventing and treating cotton verticillium wilt, wherein the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

SINA E3 ubiquitin ligase is a ubiquitous regulator that regulates plant growth and stress response at the post-translational level. The present invention first systematically identified SINA members of 24 gossypium hirsutum that contain highly conserved RING finger domains and SINA domains. The qRT-PCR method is used for analyzing that the transcription levels of GhSINA7, GhSINA8 and GhSINA9 genes are obviously up-regulated compared with uninfected control plants after cotton is inoculated with verticillium wilt bacteria for 24 hours.

The SINA gene encodes a C3HC4 type RING E3 ligase, which are normally active as dimers to maintain stability in vivo and perform various biological functions. Indeed, the RING domain spatially recruits E2, essential for the dimer formation of C3HC 4-type RING E3 ligase. RING E3 ligases of the C3HC4 type, such as SlSINA, MtSINA and MdSINA, form homo-or heterodimers with themselves or with other SINA proteins. Dimer formation needs to be dependent on the activity of RING E3 ligase in vitro. In yeast double-hybrid experiments, the proteins GhSINA7, GhSINA8 and GhSINA9 are found to interact with each other by itself or with other two GhSINAs to form homo-or heterodimers respectively. Thus, GhSINA E3 ubiquitin ligase performs ubiquitination functions by forming homo-or heterodimer complexes.

Ubiquitination is a post-translational regulatory mechanism in plants that controls a range of physiological processes. The E3 ligase can specifically recognize target protein for ubiquitination. In the invention, the inventor identifies a member SINA of RING type E3 ligase family, and researches the role of the SINA in the process of responding to verticillium wilt of upland cotton. In the experiment, 24 GhSINAs are selected, wherein the expression level of GhSINA7, GhSINA8 and GhSINA9 is obviously increased after the verticillium wilt is infected for 24 hours. All 3 GhSINAs had E3 ubiquitin ligase activity and localized to the nucleus. In addition, these GhSINAs can interact to form homodimers and heterodimers. The resistance of arabidopsis thaliana, which is ectopically overexpressed by GhSINA7, GhSINA8 and GhSINA9 genes respectively, to verticillium wilt is improved, but the resistance of cotton to pathogens is reduced by independently knocking out GhSINA7, GhSINA8 and GhSINA 9. Therefore, ghSINA7, ghSINA8 and ghSINA9 play a positive regulatory role in cotton defense against verticillium wilt.

As in some embodiments, the gene GhSINAs is gene GhSINA 7; in some embodiments, the gene GhSINAs is gene GhSINA 8; in some embodiments, the gene GhSINAs is gene GhSINA 9; in some embodiments, the gene GhSINAs is gene GhSINA7 and gene GhSINA 8; in some embodiments, the gene GhSINAs is gene GhSINA7 and gene GhSINA 9; in some embodiments, the gene GhSINAs is gene GhSINA8 and gene GhSINA 9; in some embodiments, the gene GhSINAs is gene GhSINA7, gene GhSINA8, and gene GhSINA 9.

In addition, it should be noted that, in the present invention, genes GhSINA7, GhSINA8 and GhSINA9 are present in the a subgroup chromosome and the D subgroup chromosome, respectively, and the sequences of the homologous gene pairs present on different chromosomes are highly similar, specifically, see the two nucleic acid sequences of the above genes GhSINA7, GhSINA8 and GhSINA 9. It is difficult to distinguish these homologous gene pairs by real-time fluorescent PCR (qRT-PCR), so that, in the subsequent studies of overexpression and gene knockout, a pair of primers is designed for each homologous gene to amplify.

Further, the gene GhSINAs comprises any one or more of gene GhSINA8 and gene GhSINA 9.

As in some embodiments, the gene GhSINAs is gene GhSINA 8; in some embodiments, the gene GhSINAs is gene GhSINA 9; in some embodiments, the gene GhSINAs comprises gene GhSINA8 and gene GhSINA 9.

Furthermore, the gene GhSINAs is applied to the genetic improvement or molecular breeding of cotton verticillium wilt resistance.

Namely, the gene GhSINAs of the invention can be used for the genetic improvement or molecular breeding of cotton verticillium wilt resistance.

The existing evidence shows that the expression of the gene GhSINAs in the cotton material is enhanced, and cotton seedlings with the gene GhSINAs over-expressed are obtained, and the resistance of the cotton seedlings to verticillium wilt is increased.

Specifically, in the cotton material, the sequence of an amplification primer used by an overexpression gene GhSINA7 is shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the sequences of amplification primers used by the overexpression gene GhSINA8 are shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the sequences of amplification primers used by the overexpression gene GhSINA9 are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

The invention designs the primer for over-expressing the gene GhSINAs by analyzing the gene GhSINAs, and the target gene fragment obtained by amplification is transferred into a plant body through a vector, so that the over-expressing gene GhSINAs can be effectively and stably expressed, and the anti-verticillium wilt property is obviously expressed.

The basic principle of gene overexpression is that a regulation element is added at the upstream of a target gene in an artificial construction mode, so that the gene can realize a large amount of transcription and translation under the condition of artificial control, and thus, the overexpression of a gene product is realized.

The general procedure for gene overexpression is as follows: cloning a target gene, selecting a specific vector (containing an enhanced promoter), and connecting the target gene to the specific vector; the vector is then introduced into the biological material.

Among them, the vector-mediated transformation method is a method of inserting a target gene into a vector molecule such as DNA of an Agrobacterium plasmid or virus and introducing the target gene into a plant genome with the transfer of the vector DNA. In addition, other approaches, such as germplasm systems, including pollen tube approaches, germ cell invasion, embryo sac and ovary injection; as another example, a gene direct introduction method, which is a physical or chemical method for directly introducing a foreign target gene into the genome of a plant, includes a gene gun transformation method, an electric excitation transformation method, an ultrasonic method, a microinjection method, a laser microbeam method, and the like, and chemical methods include a PEG-mediated transformation method, a liposome method, and the like.

In the present invention, the specific contents of the overexpression of the gene concerned are carried out according to the conventional procedures.

Further, the cotton material includes materials suitable for tissue culture of sexually, asexually, or regenerable cells.

In particular, the cotton material comprises any one of the following: leaf, root, stem, radicle, germ, seed.

In the above, the over-expression of the gene GhSINAs in the cotton material may be the cotton tissue in planting or growing, or the cotton material in vitro used for cotton propagation.

The invention also provides a method for detecting the verticillium wilt resistance of cotton, which detects the expression condition of the gene GhSINAs in a sample to be detected, and judges that the cotton is verticillium wilt resistant if the gene GhSINAs is over-expressed;

wherein, the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

The detection of the gene GhSINAs in the sample to be detected can be carried out in various ways, such as directly detecting whether the gene GhSINAs itself is contained or detecting products generated by the gene GhSINAs, wherein the products comprise direct products or indirect products or secondary products, and the products can be mRNA, protein, a certain compound, and the like.

Detecting the cotton verticillium wilt-resistant strain with the gene GhSINAs over-expression by a molecular biological detection means to select the cotton verticillium wilt-resistant strain.

For example, the gene GhSINAs can be directly detected by using a specific primer pair of the gene GhSINAs, or by using a probe or a chip designed for the gene GhSINAs.

Furthermore, the detection of the sample to be detected is carried out by a detection primer pair or a probe or a chip aiming at the overexpression of the gene GhSINAs.

According to the overexpression target fragment of the gene GhSINAs designed by the invention, further, the nucleic acid sequence of a detection primer pair of the gene GhSINA7 is shown as SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure;

the nucleic acid sequence of the detection primer pair of the gene GhSINA8 is shown as SEQ ID NO: 15 and SEQ ID NO: 16 is shown in the figure;

the nucleic acid sequence of the detection primer pair of the gene GhSINA9 is shown as SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

The invention also provides a method for cultivating the verticillium wilt-resistant cotton variety, which comprises the steps of preparing cotton seedlings containing the overexpression of the gene GhSINAs;

the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

Verticillium wilt is a highly invasive fungal disease that severely reduces cotton fiber quality and yield. Approximately two weeks after infestation, diseased cotton plants develop visual disease symptoms: leaf wilting, leaf chlorosis and severe growth retardation. Transgenosis, VIGS and post-infection phenotypes are effective ways to reveal molecular mechanisms for cotton gene regulation of verticillium wilt. The invention verifies that ectopic overexpression of the GhSINA7, GhSINA8 and GhSINA9 genes improves the resistance of transgenic arabidopsis thaliana to verticillium wilt bacteria, and simultaneously, preliminary evidence shows that transgenic cotton over expressing the genes also improves the resistance of transgenic arabidopsis thaliana to verticillium wilt bacteria. The gene GhSINA7, GhSINA8 and GhSINA9 are knocked out to reduce the resistance of plants to verticillium wilt. These findings indicate that the loss and gain of function of SINAs in response to a large verticillium wilt infestation is consistent with GhSINAs' resistance to verticillium wilt pathogens in plants. On this basis, we will obtain stable over-expressed and gene-edited transgenic cotton lines resistant to verticillium wilt.

Further, the cotton seedlings are obtained from a material suitable for tissue culture of sexual, vegetative or regenerable cells.

Specifically, the material suitable for tissue culture includes any one of the following: leaf, root, stem, radicle, germ, seed.

Furthermore, the sequences of amplification primers used for over-expressing the gene GhSINA7 are shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the sequences of amplification primers used by the overexpression gene GhSINA8 are shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the sequences of amplification primers used by the overexpression gene GhSINA9 are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

It should be noted that the cotton in the present invention may include various plant cottons, such as upland cotton (Gossypium hirsutum), sea island cotton (Gossypium barbadense), asian cotton (Gossypium arboreum), and grass cotton (Gossypium herbaceum), and varieties of these cotton varieties. That is, cotton varieties having the same or similar genes as the gene GhSINAs of the present invention are within the scope of the present invention. The similarity of the similar gene and the sequence of the gene GhSINAs of the invention can reach more than 80%, or the similarity can reach more than 85%, or more than 90%, or more than 95%, or more than 96%, or more than 97%, or more than 98%, or more than 99%, etc.

Compared with the prior art, the beneficial effects of the invention at least comprise the following aspects:

(1) the invention firstly defines the function of the gene GhSINAs in cotton verticillium wilt-resistant bacteria and provides a target gene for preventing and treating verticillium wilt infection of cotton.

(2) The invention obtains the overexpression of GhSINA7, GhSINA8 and GhSINA9 genes through a plurality of tests, which improves the resistance of transgenic plants to verticillium wilt; knocking out the genes GhSINA7, GhSINA8 and GhSINA9 reduces the resistance of plants to verticillium wilt bacteria, indicating that the loss and gain of function of SINAs in response to infection by verticillium wilt disease is consistent with the resistance of GhSINAs to verticillium wilt bacteria in plants.

(3) The gene GhSINAs discovered by the invention can be applied to preventing and treating cotton verticillium wilt, and particularly can be applied to the aspects of cotton verticillium wilt resistance genetic improvement or molecular breeding, such as breeding of cotton varieties with verticillium wilt resistance.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 is a diagram of the analysis of the conserved domain and developmental system of cotton SINA ubiquitin ligase of example 1;

FIG. 2 is a histogram showing the expression level of the SINA gene after inoculation of verticillium dahliae for 24 hours in the root of cotton No. 2 planted in resistant variety upland cotton in example 2 of the present invention;

FIG. 3 is a graph showing the results of the yeast two-hybrid assay for detecting the interaction between 3 GhSINA proteins (GhSINA7, GhSINA8, and GhSINA9) in example 3 of the present invention;

FIG. 4 is a graph showing the results of in vitro ubiquitination analysis of GhSINA7, GhSINA8 and GhSINA9 proteins in example 3 of the present invention;

FIG. 5 is a graph showing the results of detecting the subcellular localization of GhSINA7, GhSINA8 and GhSINA9 proteins in tobacco epidermal cells by eGFP fusion in example 3 of the present invention;

FIG. 6 is a phenotypic graph of upland cotton CLA1 gene as a positive control for silencing in example 4 of the present invention;

FIG. 7 is a bar graph of the results of the transcriptional level detection by qRT-PCR in example 4 of the present invention confirming the silencing efficiency of GhSINA7, GhSINA8 and GhSINA9 in different VIGS plants;

FIG. 8 is a bar graph showing the detection of the inhibition specificity of the genes GhSINA7, GhSINA8 and GhSINA9 in example 4 of the present invention;

FIG. 9 shows disease symptom analysis of plants silenced with GhSINA7, GhSINA8 and GhSINA9 infected with Verticillium dahliae V991 strain in example 4 of the present invention;

FIG. 10 shows the distribution of disease levels in different plants according to example 4 of the present invention;

FIG. 11 shows the vascular color changes of longitudinally cut stems of GhSINA7, GhSINA8 and GhSINA9 gene-silenced cotton plants and wild-type cotton plants after infecting verticillium wilt bacteria in example 4 of the present invention;

FIG. 12 is a graph showing the results of a fungus recovery experiment conducted on stem segments sterilized on the surfaces of control and silent plants in example 4 of the present invention;

FIG. 13 is a bar graph of the relative fungal biomass of silenced plants and control plants of example 4 of the present invention after inoculation with Verticillium dahliae;

FIG. 14 is a diagram showing the correlation between Arabidopsis plants independently overexpressing GhSINA7, GhSINA8 and GhSINA9 in example 4 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Description of materials and methods common to some of the different embodiments

1. Plant material and growth conditions

The No. 2 cotton planted in upland cotton is used as experimental material and sowed in nutritive soil in greenhouse under the conditions of 28 deg.C, 16h light/8 h dark.

The arabidopsis wild type Col-0 seeds are planted on an MS culture medium, then seedlings are transplanted into flowerpots containing nutrient soil and vermiculite, and the flowerpots are placed in a culture room at 23 ℃ under the photoperiod condition of 16h light/8 h dark.

Tobacco seedlings were cultured in a greenhouse at 25 ℃ for about 6 weeks under 16h light/8 h dark conditions.

2. Pathogenic bacteria preparation and inoculation treatment

Culturing Verticillium dahliae strain V991 in potato glucose agar (PDA) culture medium at 25 deg.C for 4 days, inoculating activated verticillium dahliae strain V991 in Czochralski culture medium at 25 deg.C and 180rpm, culturing for 5 days, and filtering with four layers of gauze. Collecting the filtered bacterial liquid, calculating by a blood counting chamber, and diluting the spore concentration to 1 x 10⁶One/ml for standby. The root of a cotton seedling cultured for two weeks under the water culture condition is inoculated with spore suspension (10ml) by adopting a root dipping method, and the spore suspension is collected for RNA extraction after 24 hours. For SINA-silenced cotton plants, the hypocotyls were injected 1 cm below the cotton cotyledons using a 1ml syringe (3. mu.l). Arabidopsis thaliana was inoculated by gently pulling 4-week-old seedlings out of the soil, carefully rinsing the roots with sterile water and soaking in a spore suspension (4X 10)⁵Conidia/ml) for 2 minutes. Then the plants were transferred to sterilized fresh soil and were continuously cultivated and observed at 23 ℃ in a greenhouse with 16h of light/8 h of dark.

3. Disease index, pathogen recovery

The disease index DI is calculated as [ (∑ disease grade × number of infected plants)/(total number of plants × 4) ] × 100. After verticillium wilt infestation, seedlings are rated on five levels, 0, 1, 2, 3, 4 and 5, depending on disease severity.

And (3) carrying out surface sterilization treatment on the control group and the SINA silent cotton plant, taking the first internode slice, cutting the first internode slice into 3-5 mm slices respectively, and culturing the slices on a PDA culture medium at 25 ℃ for 3 days. To examine the degree of multiplication of Verticillium dahliae, the longitudinal sections of cotyledonary nodes were dissected under a stereomicroscope (Leica, Wetzlar, Germany).

4. Measurement of fungal biomass

After cotton and arabidopsis are inoculated with verticillium wilt bacteria, the stems of cotton plants and the roots of arabidopsis are collected for DNA extraction. The fungus content of tissues infected with verticillium wilt bacteria of cotton and arabidopsis is detected by using an ITS1-F primer specific to fungus and a verticillium dahliae specific reverse primer STVE 1-R. The cotton Histone3 and Arabidopsis Actin2 genes were used as internal references. Real-time fluorescent quantitative PCR analysis was performed on genomic DNA.

5. RT-PCR and qRT-PCR analysis

Total RNA was extracted from different tissues of cotton or arabidopsis thaliana using TRIzol reagent (TIANGEN, beijing, china). 1. mu.g of total RNA was used for reverse transcription into cDNA (R323 version Vazyme, Nanjing, China) A. RT-PCR analysis was as described previously. The qRT-PCR detection adopts SYBR Green Real-time PCR Master Mix (Vazyme) and uses cotton Histone3 or Arabidopsis thaliana Actin2 gene as internal reference. The expression level of the target gene was measured using LightCycler480 system from Roche Germany.

Example 1

Bioinformatics analysis

To investigate the effects of cotton SINA ubiquitin ligase, 12 Asian, 12 Redmond and 24 upland cotton SINA genes were co-identified in the cotton genomic database. Of these, 3 cotton varieties, upland cotton, Asian cotton and Redmond cotton, were obtained from the Cotton FGD database (http:// www.cottonfgd.org /). Subsequently, the gossypium hirsutum SINA gene was cloned and the sequenced GhSINA8 coding sequence was modified based on its initial annotation. These SINAs encode two typically conserved domains, a RING finger domain (at the amino terminus) and a SINA domain (at the carboxy terminus) (fig. 1A)). In order to study the homologous domain sequence of Gossypium hirsutum SINA and the conserved frequency of each residue, sequence alignment was performed and the sequence characteristics of the SINA protein were obtained, as shown in FIG. 1B, the sequence identifier figure shows that the Gossypium hirsutum SINA ubiquitin ligase contains a highly conserved RING finger domain and SINA domain. In general, RING refers to a domain containing 39 conserved basic amino acid residues, while the SINA domain contains 200 conserved residues, which are responsible for interacting with a particular target substrate.

In order to further study the evolutionary relationship between cotton and Arabidopsis SINA protein, an evolutionary tree was constructed. Wherein, 5 Arabidopsis SINAs amino acid sequences (SINAT1-AT2G41980, SINAT2-AT3G58040, SINAT3-AT3G61790, SINAT4-AT4G27880 and SINAT5-A T5G53360) are searched in 3 cotton protein databases by adopting a BlastP program to screen candidate cotton SINA proteins. Candidate GhSINA proteins were then screened by Pfam (https:// www.pfam.xfam.org) and InterPro databases (https:// www.ebi.ac.uk/InterPro /) to determine whether RING finger and SINA domains were present. Phylogenetic analysis adopts MEGA 7.0 software to construct a phylogenetic tree from all SINA protein sequences of cotton and Arabidopsis by a neighborhood joining method (NJ) with 1000 bootstraps repeated, and the number on the branch represents a guide value. RING finger and SINA conserved domain protein sequences of Gossypium hirsutum SINAs were aligned using ClustalX 2.0 software. And uploading the alignment result to an online tool WEBLOGO (http:// webago. bergelley. edu/logo. cgi) to analyze the specific amino acid characteristics.

All identified SINA proteins were divided into two subgroups (FIG. 1C). The larger subgroup contained 32 cotton SINA genes (8, 8 and 16 from Raymond, Asian and upland cotton, respectively). The number of the SINA genes of upland cotton is twice that of diploid cotton Asian cotton (AA) and Raymond cotton (DD). This phenomenon is consistent with the results of the heterotetraploid upland cotton (AADD) event producing polyploids derived from the natural crossing of the diploid precursors asian cotton and raymond cotton. The second group consisted of 2 AtSINATs and 16 SINAs (4, 4 and 8 in Ramond, Asian and upland cotton, respectively), indicating that the SINA gene family was significantly expanded during the evolution of the cotton genome.

Example 2

Expression analysis of GhSINA gene response to verticillium wilt

After 24h of infecting upland cotton by verticillium wilt marked by GFP, detecting that conidia on cotton seedling roots germinate and the growth of verticillium hyphae extends. In order to determine whether the expression level of the GhSINA gene changes 24h after the verticillium wilt is infected, the transcription level of the GhSINA gene in the root system of a upland cotton resistant variety (China plant cotton No. 2) is studied, and the related primers are shown in a table 1. Among them, ghSINA7, ghSINA8 and ghSINA9 were present in the A subgroup chromosome and the D subgroup chromosome, respectively, but the sequences of their homologous gene pairs were highly similar, and it was difficult to distinguish the homologous gene pairs by real-time fluorescence PCR (qRT-PCR), and therefore, they were amplified together. FIG. 2 shows the expression level of SINA gene in resistant variety, cotton No. 2, inoculated with Verticillium dahliae at root for 24h, total RNA was extracted from root after 14 days old seedlings were inoculated with Verticillium dahliae for 24h, error bars represent three biological replicates, cotton Histone3 was used as the reference gene, asterisks indicate that the differences are statistically significant, and significant differences were analyzed with Student's t-test (. about.P < 0.01). As can be seen from FIG. 2, the expression levels of the GhSINA7, GhSINA8 and GhSINA9 genes were significantly up-regulated at 24h after inoculation and were induced approximately 3.5, 3.2 and 2 times, respectively, compared to the control group, and in general, the GhSINAs gene transcript levels were higher than those of the control group. Therefore, the regulation of verticillium wilt by the GhSINA7, GhSINA8 and GhSINA9 genes of upland cotton is intensively studied later.

TABLE 1 qRT-PCR related primers for different GhSINA genes

Example 3

1. Yeast two-hybrid experiments

Yeast two-hybrid screens were constructed according to the instructions of the Matchmaker Gold yeast two-hybrid System (Clontech, Palo Alto, Calif., USA). GhSINAs full-length cDNAs (see Table 2 for specific primers) were cloned into the bait vector pGBDK7 and prey vector pGADT7, respectively. The constructed recombinant plasmids were co-transformed into yeast Y2HGold and cultured on SD/-Leu/-Trp DO (DDO) medium at 30 ℃ for 3 days. Independent colonies of the same size were picked and transferred to SD/-Leu/Trp/-Ade/-His DO (QDO) medium containing X- α -Gal (Clontech), and the interaction between GhSINA proteins was evaluated by whether the colonies turned blue or not.

TABLE 2 GhSINAs primers for different vectors

SINA proteins perform biological functions by forming homodimers or heterodimers. To determine whether these three candidate GhSINAs proteins have the ability to form homo-or heterodimers, yeast two-hybrid experiments identified the interaction between GhSINA7, GhSINA8, and GhSINA 9. Full-length cDNAs of the GhSINA7, GhSINA8 and GhSINA9 genes were independently fused to either a DNA Binding Domain (BD) containing bait vector pGBKT7 or an Activation Domain (AD) containing prevector pGADT7, and then co-transformed into Y2HGold, and their interaction was preliminarily identified by growth on defective media. As shown in FIG. 3, the transformed yeast cells were grown in SD/-Leu/-Trp DO (DDO) medium and SD/-Leu/-Trp/-Ade/-His DO (QDO) medium (containing X-. alpha. -gal), i.e., each GhSINA interacted with itself and the other two GhSINAs to form homologous or heterologous complexes, respectively. The effectiveness of the Y2H experiment was verified by the interaction of AD-T and BD-53 as a positive control.

2. In vitro ubiquitination assay

Full-length Open Reading Frames (ORFs) of GhSINA7, GhSINA8 and GhSINA9 genes were independently cloned (primers shown in Table 3) into pMAL-C2x vector to generate fusion expression vector with Maltose Binding Protein (MBP). The recombinant plasmid was expressed in E.coli BL21 strain, purified using amylose resin (NEB, Ipswich, MA, USA) affinity chromatography, and used for in vitro ubiquitination analysis as described previously. Purified fusion proteins MBP-SINAs (3. mu.g) were incubated in 30. mu.l ubiquitination reaction buffer (50mM Tris-HCl, pH 7.5, 5mM MgCl2, 2mM DTT and 2mM ATP), 5. mu.g biotin-tagged ubiquitin (Enzo, # BML-UW9920-0001),100ng E1(Enzo,170# BML-UW9920-0001) and 40ng Human E2(UbcH5b) for 3h (Enzo,171# BML-UW9920-0001) independently at 30 ℃. The reaction was stopped by adding 5 Xsample buffer and half of the mixture was separated on a 7.5% SDS-PAGE gel. Western blotting identification was performed using anti-biotin antibody (cell signal 1:3000 dilution). And then developed on a Tanon-5200 chemiluminescent imaging system (AI600 UV, USA).

TABLE 3 ORF cloning primers for GhSINA7, GhSINA8, and GhSINA9 genes

The E3 ligase binds to E2 ubiquitin conjugating enzyme and has self-ubiquitination functional enzyme activity. To determine whether ghSINA7, ghSINA8, and ghSINA9 proteins have E3 ligase activity. Constructing SINAs expression vector containing MBPs label in colibacillus BL21 strain, purifying its expression to obtain fusion protein MBP-SINAs. Subsequent ubiquitination activity assays were performed and high molecular weight multimeric bands of ubiquitin could be detected using biotin antibodies (anti-biotin) in the presence of human E1(UBA1), E2(UbcH5b), biotinylated ubiquitin (Bt-Ub), and MBP-SINAs (FIG. 4A-C, column 1), indicating that MBP-SINAs are ubiquitinated. There was no polyubiquitination signal when MBP was substituted for MBP-SINAs, or when E1, E2, or biotin-labeled ubiquitin were absent (FIG. 4A-C, columns 2-6). It is clear that ghSINA8 has stronger E3 ligase activity than either ghSINA7 or ghSINA 9. These results indicate that all three SINA proteins contain E3 ubiquitin ligase activity. GhSINA protein is a functional E3 ubiquitin ligase.

In FIG. 4, A-C shows the E3 ubiquitin ligase activity of MBP-SINA7(A) MBP-SINA8(B) and MBP-SNA9(C) fusion proteins detected in the presence of E1, E2 and biotinylated ubiquitin (Bt-Ub) (column 1), samples were separated by 7.5% SDS-PAGE and detected using an anti-biotin antibody Western Blotting (WB), MBP was used as a negative control, and an equal amount of E3 enzyme was present in a Coomassie blue staining reaction.

3. Subcellular localization of tobacco leaves

In the modified pCambia2300-eGFP expression vector, the open frames of the GhSINA7, GhSINA8 and GhSINA9 genes were fused with eGFP, and the primers involved in GhSINA7, GhSINA8 and GhSINA9 are shown in Table 4. The bacterial liquid of agrobacterium containing recombinant plasmid (strain GV3101) is injected and permeated into tobacco leaf. The cells were incubated at 25 ℃ for 48 hours in the dark, and the fluorescence signal was observed with a confocal laser microscope (Olympus, germany) at a scale bar of 20 μm.

TABLE 4 amplification primers for GhSINA7, GhSINA8, and GhSINA9 involved in subcellular localization

Ubiquitination usually occurs in the nucleus and cytoplasm, controlling nuclear and cytoplasmic proteins, respectively, to exert biological functions. To determine the subcellular localization of ghSINA7, ghSINA8, and ghSINA9 proteins, a fusion construct of ghSINA7/8/9-eGFP, induced in the CaMV 35S constitutive promoter, was transiently expressed in tobacco leaves, respectively. As shown in fig. 5, free eGFP green fluorescence was observed in the nucleus. Apparently, these three SINA proteins are present in the nucleus where GhSINA proteins are localized, consistent with their function in the ubiquitination pathway.

Example 4

1. Silencing of GhSINAs gene reduces cotton resistance to verticillium wilt

(1) Cotton Virus Induced Gene Silencing (VIGS)

pTRV1 and pTRV2 vectors were used in VIGS experiments. Specific fragments of GhSINAs (primers shown in table 5) were amplified and ligated into pTRV2 vector. The recombinant plasmid was transformed into Agrobacterium GV 3101. Subsequently, agrobacterium inoculum (OD600 ═ 1) containing pTRV1 and pTRV2-GhSINA plasmid was mixed at a ratio of 1:1, and the inoculum was injected into two whole cotyledons of 7-day-old cotton seedlings using a syringe.

TABLE 5 amplification primers for silencing genes GhSINA7, GhSINA8, and GhSINA9

(2) Results

An effective method for gene silencing in a wide range of applications in cotton research is VIGS. The VIGS technology is adopted to research the function of the GhSINAs gene in regulating and controlling cotton verticillium wilt resistance. Therefore, VIGS-specific silencing of these three GhSINA genes (TRV2: GhSINA7, TRV2: GhSINA8 and TRV2: GhSINA9) was used to investigate their function in responding to verticillium wilt. The positive and negative controls were GhCLA1 and TRV 00 blank, which gave a markedly albino phenotype upon silencing. After the agrobacteria solution permeated the cotton leaves for 7 days, the cotton leaves showed the expected albino phenotype after the CLA1 gene was silenced (fig. 6), which indicates that the VIGS experiment performed well. The expression level of the silenced gene in different silenced strains is detected respectively (the related primers are shown as GhSINA7, GhSINA8 and GhSINA9 in Table 1). Expression levels of ghSINA7, ghSINA8 and ghSINA9 genes were significantly reduced in VIGS plants compared to TRV:00 treated plants, respectively (FIG. 7). To investigate the VIGS-mediated specificity of three SINAs for inhibition of genes GhSINA7, GhSINA8, and GhSINA9, the transcript levels of two additional non-targeted SINA genes with high similarity to the silenced GhSINA coding sequence in each gene-silenced plant were examined. The expression levels of both non-targeted SINA genes were not affected in each specifically silenced plant (fig. 8).

Subsequently, the control plants and the silenced plants were infected with verticillium wilt. After about two weeks, genetically silenced cotton seedlings, especially those containing TRV2: GhSINA8 and TRV2: GhSINA9, showed more severe leaf withering, yellowing and defoliation symptoms and even death (TRV:00) (FIG. 9). Counting DIs, most TRV2: GhSINA8 and TRV2: GhSINA9 plants have more serious disease lesions (figure 10), in figure 10, 0 grade is healthy leaves; grade 1, 0 to 25 percent of leaves wither; and 2, stage: 25% -50% withered or chlorosis in the leaves; and 3, level: 50% -75% of the leaf is chlorosis or necrosis; 4, level: the leaves died or fell off, the experiment was performed in 3 replicates, with at least 40 seedlings in each replicate, and the asterisks indicate that the differences were statistically significant as judged by rank test (P <0.05, P < 0.01). Furthermore, GhSINA7, GhSINA8 and GhSINA9 gene silenced cotton plants showed darker vascular color than wild type cotton plant stalks, especially more necrotic vascular tissue in GhSINA8 and GhSINA9 silenced cotton plants as compared to TRV:00 (fig. 11). Fungal recovery experiments demonstrated that plants silenced with GhSINA8 and GhSINA9 had more fungal growth than the control (fig. 12). Accordingly, the fungal biomass of GhSINA8 and GhSINA9 silenced plants was significantly higher than control plants (fig. 13). Figure 13 compares DNA levels between the verticillium wilt pathogen Internal Transcribed Spacer (ITS) and cotton Histone3 by qRT-PCR analysis, with V values representing the mean ± SDs of three biological replicates (. about.p <0.01, Student's t test). In conclusion, silencing GhSINA7, GhSINA8 and GhSINA9 can inhibit the immune system of plants and enhance the susceptibility of plants to verticillium wilt.

2. Overexpression of GhSINAs gene improves resistance of transgenic arabidopsis to verticillium wilt

(1) Plasmid construction and Arabidopsis transformation

The open frame of GhSINAs (primers involved are shown in Table 6) was cloned and inserted independently into the vector pCambia2300(CAMBIA) containing the CaMV 35S constitutive promoter. All constructs were sequenced and confirmed and introduced into Agrobacterium tumefaciens GV3101, respectively. The Columbia wild type Arabidopsis thaliana was transformed by Agrobacterium tumefaciens-mediated inflorescence dip-staining. Transgenic arabidopsis thaliana was screened on MS medium containing kanamycin, and the selected transgenic seedlings were further screened using genomic PCR technology. The homozygous T3 transgenic line was obtained for functional analysis.

TABLE 6 amplification primers for genes involved in overexpression of GhSINA7, GhSINA8, and GhSINA9

(2) Results

A gene overexpression strategy is adopted to evaluate the function of the GhSINAs gene in defense response. The model plant Arabidopsis thaliana was used as the test material in this experiment because of the longer cotton transformation period. During this time we obtained more than 18 independently overexpressed ghSINA7, ghSINA8, and ghSINA9 transgenic Arabidopsis lines. Two independent homozygous T3 lines with highest SINA expression level (primers shown in Table 7) were selected for phenotypic analysis (FIG. 14A), FIG. 14A identifies transgenic Arabidopsis lines independently overexpressing GhSINA7, GhSINA8 and GhSINA9 by RT-PCR, and Arabidopsis Actin2 is used as an internal control. After 4 weeks of planting, transgenic plants overexpressing SINAs were inoculated with verticillium wilt for resistance determination. Two weeks after inoculation, the plants began to develop disease symptoms and, overall, the transgenic plants showed stronger resistance to leaf expression than wild-type plants (fig. 14B), fig. 14B is a plot of symptoms after inoculation of verticillium wilt in wild-type and GhSINA7, GhSINA8, GhSINA9 transgenic plants. The disease resistance of the GhSINA7 transgenic line is improved compared with the wild type, but is far lower than that of GhSINA8 and GhSINA9 transgenic lines. FIG. 14C shows the disease rate statistics of necrotic leaves of wild-type and transgenic Arabidopsis plants, error bars represent 3 biological replicates (n.gtoreq.32), asterisks indicate that the differences are statistically significant, and significant differences were analyzed by Student's t-test (P <0.05, P <0.01), and it can be seen that the necrosis rate of transgenic lines of GhSINAs is significantly lower than that of WT (FIG. 14C). In addition, FIG. 14D is a bar graph of the results of quantitative measurements of fungal biomass using qRT-PCR analysis to compare the transcript levels between the Internal Transcribed Spacer (ITS) of Verticillium wilt bacteria and the Arabidopsis thaliana Actin2 gene, where the asterisks indicate that the differences are statistically significant and the significant differences were analyzed using Student's t-test (P <0.05, P < 0.01). Fungal biomass experiments demonstrated that less fungal DNA was accumulated in the root system of transgenic plants, especially in GhSINA8 and GhSINA9 overexpressing lines (FIG. 14D). Therefore, ectopic overexpression of GhSINAs greatly enhances the resistance of Arabidopsis thaliana to verticillium dahliae compared with the wild type.

TABLE 7 primers involved in overexpression detection

Wherein, in the research that the silencing of GhSINAs reduces the resistance of cotton to verticillium wilt and the overexpression of GhSINAs improves the resistance of transgenic arabidopsis to verticillium wilt, the primers used for detecting the fungal biomass are shown in Table 8.

TABLE 8 primers used for fungal Biomass detection

Primer name	Primer and method for producing the same
		ITS1-F	AAAGTTTTAATGGTTCGCTAAGA
STVE1-R	CTTGGTCATTTAGAGGAAGTAA

In addition, transgenic cotton lines that independently overexpress GhSINA7, GhSINA8, and GhSINA9, were initially shown to have verticillium wilt resistance consistent with arabidopsis thaliana.

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Sequence listing

<110> Cotton research institute of Chinese academy of agricultural sciences

Application of <120> gene GhSINAs in prevention and treatment of cotton verticillium wilt

<130> 2021

<160> 18

<170> SIPOSequenceListing 1.0

<210> 1

<211> 915

<212> DNA

<213> G．hirsutum

<400> 1

atggcatctg ggaatccttt ttttgatgac ttgcggagca agcctgaggt tattgatctt 60

ccacagaacg aagaccgttt ggatagtgag tccgtgaacg aacctactca aactacaaca 120

aaaccgaatt tgattgtgtc tagcaatgtt cgtgaactgt tggaatgccc tgtttgctta 180

aatgcgatgt acccaccaat tcatcagtgc ctaaatgggc acacgttgtg ttctggttgc 240

aagccaagag tgcacaatcg gtgccccaca tgcagacatg agcttgggaa tattagatgt 300

cttgcattgg agaaggtcgc tgcatctctt gagcttccct gtaaatatca aagttatggg 360

tgcgtaggca tatatcccta ttacagcaaa ctaaaacatg agtcccagtg ttcatataga 420

ccatatagtt gcccttatgc tgggtcagaa tgcacagtgg ttggtgatat tccttttctt 480

gtggctcacc tgaaagatga tcacaaagtt gacatgcaca gtggcagcac cttcaaccat 540

cgttatgtca aatcaaatcc gcatgaggtt gaaaatgcta catggatgtt gacggttttc 600

agttgctttg gtcagtactt ttgcctgcat ttcgaagcct tccaacttgg gatggctccc 660

gtttatatag cattcttgcg gtttatgggt gatgataatg aggcaaagaa ctacagctac 720

agccttgagg tgggtgggaa tgggaggaag ttgatttggc aaggggttcc aagaagcata 780

cgagacagtc atcgaaaggt tcgtgacagt ttcgacggtc tcatcatcca acgcaacatg 840

gcattgttct tctcgggagg agacagaaag gagttgaagc ttagagtaac aggaaggata 900

tggaaagagc agtga 915

<210> 2

<211> 915

<212> DNA

<213> G．hirsutum

<400> 2

atggcatctg ggaatccttt ttttgatgac ttgcggagca agcctgaggt tattgatctt 60

ccacagaacg aagaccgttt ggatagtgag tccgtgaatg aacctactca aactgcaaca 120

aaaccgaatt tgattgtgtc tagcaatgtt cgtgaactgt tggaatgccc tgtttgctta 180

aatgcaatgt acccaccaat tcatcagtgc ctaaatgggc acacattgtg ttctggttgc 240

aagccaagag tgcacaatcg gtgccccaca tgcaggcatg agcttgggaa tattagatgt 300

cttgcattgg agaaggtcgc tgcatctctt gagcttccct gtaaatatca aagttatggg 360

tgcgtaggca tatatcccta ttacagcaaa ctaaaacatg agtcccagtg ttcgtataga 420

ccatatagtt gcccttacgc tgggtcagaa tgcacagtcg ttggtgatat tccttttctt 480

gtggctcacc tgaaagatga tcacaaagtt gacatgcaca gtggcagcac ctttaaccat 540

cgttatgtca aatcaaatcc gcatgaggtt ggaaatgcta catggatgtt gacggttttc 600

agttgctttg gtcagtactt ttgcctgcat ttcgaagcct tccaacttgg gatggctcct 660

gtttatatag cattcttgcg gtttatgggt gatgatgacg aggcaaagaa ctacagctac 720

agccttgagg tgggtgggaa tgggaggaag ttgatttggc aaggggttcc aaggagcata 780

cgagacagtc atcgaaaggt tcgtgacagt tttgatggtc tcatcatcca acgcaacatg 840

gcattgttct tctcgggagg agacagaaag gagttgaagc ttagagtaac aggaaggata 900

tggaaagagc agtga 915

<210> 3

<211> 1011

<212> DNA

<213> G．hirsutum

<400> 3

atggagtcgg atactatcga gtgcgtatca tctatagatg aaattgttga agatcatgag 60

atccctcacc ataatctact ccaccaccca caccctcgtc atgcccctca tcatcaattt 120

tcatcgtcaa agcctcatca caacggcact aataatgtta atagtattag caatatcgtg 180

ggtccaacgg cgattgctcc ggctgctagt gtccatgaat tgttagaatg tcctgtttgc 240

accaattcta tgtatccacc aattcatcag tgccacaatg gacatacact ttgttctacc 300

tgtaaaatac gagtacacaa ccggtgccca acttgtcggc aggaactcgg tgatattaga 360

tgtttagcat tggagaaagt tgccgagtcc cttgagttac cttgcaaata ctacaagttg 420

gggtgcccgg aaacattccc ctattacagc aaactcaaac acgaggggat ttgcatctac 480

agaccataca attgtccata tgctggttca gagtgttccg ttgttgggga tatcccattc 540

cttgttgccc atttgaggga tgatcataag gtcgacatgc acaccggctg cacatttaat 600

catcgatacg tgaaatccaa ccctcgggaa gttgaaaatg ctacatggat gctaacagta 660

ttccattgtt ttggtcagta tttctgccta cactttgaag ccttccagtt gggaatggct 720

ccagtttaca tggcatttat ccgtttcatg ggtgacgaga cggaagctag gaactatagc 780

tacagccttg aagtcggagc aaacggacgg aaacttatat gggaaagcgc cccacgtagc 840

atccgtgata gtcaccggaa agtcagggac agccatgatg gtctaattat ccaacgaaac 900

atggcacttt tcttctccgg tggggataga aaggagctga aactgagggt tactggacga 960

atatggaagg aacaacaaaa tccagatgcc aacatgtgca taccaaacta a 1011

<210> 4

<211> 1011

<212> DNA

<213> G．hirsutum

<400> 4

atggagtcgg atagtatcga gtgcgtgtca tctatagatg aaattgttga agatcatgag 60

atccttcacc ataatataca gcaccaccca caccctcgtc atgcccccca tcatcaattc 120

tcatcgtcaa agcctcatca caacggcact aataatgtta atagtattag caatatcgtg 180

ggtccaacgg cgattgctcc ggctgctagt gtccatgaat tgttagaatg tcctgtttgt 240

accaattcta tgtatccacc aattcatcag tgccacaatg gacatacact ttgttctacc 300

tgtaaaatac gagtacacaa ccggtgcccg acttgtcggc aggaactcag tgatattaga 360

tgtttagcat tggagaaagt cgccgagtcc ctcgagttac cttgcaaata ctacaagttg 420

gggtgcccgg aaatattccc ctattacagc aaactcaaac acgaggggat ttgcatctac 480

agaccataca attgtccata tgctggttcg gagtgttctg ttgttgggga tatccctttc 540

cttgttgccc atttgaggga tgatcataag gtcgacatgc acaccggctg cacatttaat 600

catcgatacg tgaaatccaa ccctcgggaa gtcgaaaatg ctacatggat gctaacagtt 660

ttccattgtt ttggtcagta tttctgccta cactttgaag ccttccagtt gggaatggct 720

ccagtttaca tggcatttat ccgtttcatg ggtgacgaga cggaagctag gaactatagc 780

tacagccttg aagtcggagc aaacggccgg aaacttatat gggaaggcac cccacgtagc 840

atccgtgata gtcaccggaa agtcagggac agccacgatg gtctaattat ccaacgaaac 900

atggcacttt tcttctccgg tggggataga aaggagctga aactgagggt tactggacga 960

atatggaagg aacaacaaaa tccagatgcc aacatgtgca taccaaacta a 1011

<210> 5

<211> 972

<212> DNA

<213> G．hirsutum

<400> 5

atggaatttg atagcattga ttgtatgaca tcttcagatg tgattgatga tgatgatgag 60

atccatcatc ataatcagtt gtcttcattg ttgaagtccc atagcaacaa tgggagcagc 120

aacagtattg tgtcacctgc ggttcactca agcaccacca gtgtccatga acttctggaa 180

tgtcctgttt gtaccaattc tatgtaccct cccatccatc agtgtcacaa tgggcatact 240

ctctgttcga cctgtaaaac aagggtacac aatcggtgcc ccacttgtag acaggagctt 300

ggtgatatta ggtgtctagc actagagaag gtagctgaat cacttgaact gccttgcaaa 360

tatacatcac ttggatgccc ggagatcttt ccttactaca gtaaactcaa acatgaggcc 420

ctatgcaact tcaggccata caattgccca tatgctggat cagaatgcac tgttgttggt 480

ggtattccat tccttgttgc tcatctaagg gatgaccaca aggttgacat gcattctggt 540

tgcacattta accatcgtta tgtgaagtcc aatcctcggg aagtagaaaa tgccacatgg 600

atgctaactg tattccactg ttatggccag tacttctgtc tccattttga agccttccag 660

cttgggatgg cccctgttta tatggcattc cttcgtttca tgggtgatga ggttgaatcc 720

cgcaactaca gttatagcct agaagttggg ggcaatggca ggaaactcat ttgggaaggc 780

actccaagaa gcataagaga tagccaccga aaggtcaggg atagccatga cggccttatt 840

atacagcgta acatggcact tttcttctct ggaggagata ggaaagagtt gaagctgcga 900

gtaaccggac ggatatggaa agaacaacag aacccagaag gtggtgcctg catacccaac 960

ctctgcagtt aa 972

<210> 6

<211> 970

<212> DNA

<213> G．hirsutum

<400> 6

atggaatttg atagcattga ttgtatgaca tcttcagatg tgattgatga tgatgatgag 60

atccatcatc ataatcagtt gtcttcattg ttgaagtccc atagcaacaa tgggagcagc 120

aacagtattg tgtcacctgc ggttcactca agcaccacca gtgtccatga aaacaagggt 180

acacaatcgg accaattcta tgtaccctcc catccatcag tgtcacaatg ggcatactct 240

ctgttcgacc tgtaaaacaa gggtacacaa tcggtgcccc acttgtagac aggagcttgg 300

tgatattagg tgtctagcac tagagaaggt agctgaatca cttgaactgc cttgcaaata 360

tacatcactt ggatgcccgg agatctttcc ttactacagt aaactcaaac atgaggccct 420

atgcaacttc aggccataca attgcccata tgctggatca gaatgcactg ttgttggtgg 480

tattccattc cttgttgctc atctaaggga tgaccacaag gttgacatgc attctggatg 540

cacgtttaac catcgttatg tgaagtccaa tcctcgggaa gtagaaaatg ccacatggat 600

gctaactgta ttccactgtt atggccagta cttctgtctc cattttgaag ccttccagct 660

tgggatggcc cctgtttata tggcattcct tcgtttcatg ggtgatgagg ttgaatcccg 720

caactacagt tatagcctag aagttggggg caatggcagg aaactcattt gggaaggcac 780

tccaagaagc ataagagata gccaccgaaa ggtcagggat agccatgacg gccttattat 840

acagcgtaac atggcacttt tcttctccgg aggagatagg aaagagttga agctgcgagt 900

aaccggacgg atatggaaag aacaacagaa cccagaaggt ggtgcctgca tacccaacct 960

ctgcagttaa 970

<210> 7

<211> 47

<212> DNA

<213> Artificial sequence ()

<400> 7

cacgggggac tcttgaggat ccatggcatc tgggaatcct ttttttg 47

<210> 8

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 8

ctagctttgt atatcactgc agtcactgct ctttccatat ccttcc 46

<210> 9

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 9

cacgggggac tcttgaggat ccatggagtc ggatactatc gagtg 45

<210> 10

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 10

ctagctttgt atatcactgc agttagtttg gtatgcacat gttggc 46

<210> 11

<211> 46

<212> DNA

<213> Artificial sequence ()

<400> 11

cacgggggac tcttgaggat ccatggaatt tgatagcatt gattgt 46

<210> 12

<211> 45

<212> DNA

<213> Artificial sequence ()

<400> 12

ctagctttgt atatcactgc agttaactgc agaggttggg tatgc 45

<210> 13

<211> 20

<212> DNA

<213> Artificial sequence ()

<400> 13

cttccacaga acgaagaccg 20

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 14

attctgaccc agcataaggg c 21

<210> 15

<211> 19

<212> DNA

<213> Artificial sequence ()

<400> 15

tgctccggct gctagtgtc 19

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 16

cagccggtgt gcatgtcg 18

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence ()

<400> 17

gcaccaccag tgtccatgaa c 21

<210> 18

<211> 22

<212> DNA

<213> Artificial sequence ()

<400> 18

gcaaccagaa tgcatgtcaa cc 22

Claims (10)

1. The application of gene GhSINAs in preventing and treating cotton verticillium wilt, wherein the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

2. The use of claim 1, wherein said gene GhSINAs comprises any one or more of gene GhSINA8 and gene GhSINA 9.

3. The use of claim 1, wherein the gene GhSINAs is used for the genetic improvement or molecular breeding of cotton against verticillium wilt;

further, the expression of the gene GhSINAs in the cotton material is enhanced, and cotton seedlings with the gene GhSINAs over-expressed are obtained, and the resistance of the cotton seedlings to verticillium wilt is increased.

4. The use of claim 3, wherein in the cotton material, the amplification primer sequence used for over-expressing gene GhSINA7 is shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the sequences of amplification primers used by the overexpression gene GhSINA8 are shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the sequences of amplification primers used by the overexpression gene GhSINA9 are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

5. The use of claim 4, wherein the cotton material comprises a material suitable for tissue culture of sexually, asexually or regenerable cells;

further, the cotton material comprises any one of the following: leaf, root, stem, radicle, germ, seed.

6. The method for detecting the verticillium wilt resistance of cotton is characterized in that the expression condition of the gene GhSINAs in a sample to be detected is detected, and if the gene GhSINAs is over-expressed, the verticillium wilt resistance of the cotton is judged;

wherein, the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

7. The detection method according to claim 6, wherein the sample to be detected is detected by a detection primer pair or a probe or a chip for overexpression of the gene GhSINAs;

further, the nucleic acid sequence of the detection primer pair of the gene GhSINA7 is shown as SEQ ID NO: 13 and SEQ ID NO: 14 is shown in the figure;

the nucleic acid sequence of the detection primer pair of the gene GhSINA8 is shown as SEQ ID NO: 15 and SEQ ID NO: 16 is shown in the figure;

the nucleic acid sequence of the detection primer pair of the gene GhSINA9 is shown as SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

8. A method for cultivating a cotton variety with verticillium wilt resistance is characterized in that cotton seedlings containing gene GhSINAs overexpression are prepared;

the gene GhSINAs comprises any one or more of gene GhSINA7, gene GhSINA8 and gene GhSINA 9;

the nucleic acid sequence of the gene GhSINA7 is shown as SEQ ID NO. 1 and/or SEQ ID NO. 2;

the nucleic acid sequence of the gene GhSINA8 is shown as SEQ ID NO. 3 and/or SEQ ID NO. 4;

the nucleic acid sequence of the gene GhSINA9 is shown in SEQ ID NO. 5 and/or SEQ ID NO. 6.

9. The method of claim 8, wherein the cotton seedlings are obtained from material suitable for tissue culture of sexual, vegetative or regenerable cells;

further, suitable materials for tissue culture include any of the following: leaf, root, stem, radicle, germ, seed.

10. The method as claimed in claim 8 or 9, wherein the amplification primer sequence for overexpression of gene GhSINA7 is shown as SEQ ID NO. 7 and SEQ ID NO. 8;

the sequences of amplification primers used by the overexpression gene GhSINA8 are shown as SEQ ID NO. 9 and SEQ ID NO. 10;

the sequences of amplification primers used by the overexpression gene GhSINA9 are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

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