CN117587047A

CN117587047A - Application of GhMPK9 gene in improving verticillium wilt resistance of cotton

Info

Publication number: CN117587047A
Application number: CN202311382890.2A
Authority: CN
Inventors: 郭旺珍; 米新月; 李维希
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2023-10-24
Filing date: 2023-10-24
Publication date: 2024-02-23

Abstract

The invention discloses application of GhMPK9 gene in improving verticillium wilt resistance of cotton, and belongs to the field of biotechnology application. The invention relates to a GhMPK9 gene which codes for a protein kinase. The invention provides a full-length ORF nucleotide sequence and an amino acid sequence of GhMPK9 in upland cotton genetic standard line TM-1. The gene is expressed in all tissues of cotton and is obviously up-regulated by the induction of verticillium wilt. GhMPK9 is a key gene for cotton reproductive development, and the expression of the gene is inhibited in cotton to influence the cotton plant reproductive development, so that offspring cannot be produced. The over-expression of the gene in cotton activates immune response, obviously improves plant disease resistance, but does not affect normal growth and development of plants.

Description

Application of GhMPK9 gene in improving verticillium wilt resistance of cotton

Technical Field

The invention belongs to the field of biotechnology application, and relates to application of a cotton GhMPK9 gene in improving verticillium wilt resistance of cotton, wherein the gene codes for a mitogen activated protein kinase. Through identification and analysis of cotton MAPK families, 28 MAPK genes are identified in total in upland cotton genetic standard line TM-1, one verticillium wilt-resistant key gene GhMPK9 is screened, full-length sequence cloning and interaction verification are further carried out, the functions of GhMPK9 in cotton verticillium wilt resistance are determined by combining transgene verification and molecular mechanism analysis, key disease-resistant genes GhRAF39_1 and GhWRKY40a at the downstream of GhMPK9 are excavated, and the GhRAF39_1 is found to play an important role in mediating GhMPK9 phosphorylation GhWRKY40a to regulate cotton verticillium wilt resistance. The GhMPK9 gene is conserved in cotton, with 2 copies in tetraploid cotton, one in each A, D subgroup. GhMPK9 has higher expression in cotton root, stem and flower organs, is obviously up-regulated by verticillium induction and has highest expression level at 12 hours. The full-length ORF sequence and the coded amino acid sequence of the GhMPK9 gene A subgroup are obtained in upland cotton by utilizing a PCR technology. The biological technology is utilized to specifically research the disease resistance and the functional mechanism of the plant participated by the gene, ghMPK9 is a key gene for cotton growth and development, and the gene is knocked out in cotton to influence the development of stamens and pistils of the plant, obviously interfere the reproductive development of the plant and can not form bell subculture. The over-expression of the gene in cotton activates the immune response of cotton, and the disease resistance of plants is obviously improved.

Background

Verticillium is a soil-borne semi-living nutritive fungus which can infect more than 200 dicotyledonous plants, including cotton, tomatoes, cucumbers, peppers, eggplants, potatoes and other main cash crops, and has the characteristics of wide host range, high transmission speed, long survival time in soil and the like, and is difficult to control in fields (Bhat and Subbarao, 1999). The life cycle of verticillium begins with germination of microsclerotia, which begins to produce germ tubes and extends longitudinally along root epidermal cells after the secretion released by the plant root is perceived as a signal. Wherein a small amount of hypha top is swelled and is tightly adsorbed on the root surface in the form of attaching branches, so that a narrow infection nail structure is formed to penetrate the epidermis for infection; in the infection process, pathogenic bacteria secrete some jelly to firmly fix the attached branches on the infection interface so as to bear the recoil force generated at the moment of penetration; simultaneously releasing large amounts of cell wall hydrolases, such as pectinases, cutinases, cellulases, and the like, to destroy plant cell wall tissue (Sesma and Osbourn,2004; zhao et al, 2014; zhao et al, 2016); a number of effectors disrupt or interfere with the immune response of plants (de Jonge et al 2011). The mycelium after successful invasion gradually spreads to a central location, eventually enters the xylem and colonizes, grows and breeds in or between cells, and spreads up the xylem vessel to the stem or leaf (shaman et al, 2018). Cotton is one of the most important crops in the world, is not only the most important fiber crop, but also important oil crop, is also a high-protein-containing grain crop, and has remarkable comprehensive utilization value. In recent years, verticillium wilt becomes one of the main obstacles in the production of high-yield and high-quality cotton in China due to the change of climate conditions, a long-term single-cropping planting mode and a new cotton variety which is frequently introduced. The verticillium wilt is a first major disease of cotton, and has wide transmission path, and various mediums such as cotton seeds, plant residues, soil, fat water, farm tools and the like can be transmitted, so that the disease is serious, the leaves of light people lose green and turn yellow, the shedding of the cotton bolls seriously reduces the yield, the whole plant of heavy people die in a piece, and the yield is absolutely kept. The chemical control strategy for verticillium wilt is not ideal, and management means such as field rotation and the like can only properly relieve the occurrence of diseases, so that the method for excavating key disease-resistant genes in cotton and creating disease-resistant materials is the most direct and effective strategy for improving disease resistance.

Plants and pathogenic bacteria struggle with each other and also exhibit various means and abilities to fight against pathogens during co-evolution. During the last decade, researchers have conducted a series of studies on the physiological and molecular mechanisms by which plants resist vascular bundle disease caused by verticillium dahliae. It has now been found that plants can develop resistance to verticillium by a variety of regulatory mechanisms, including cell wall modification, extracellular enzyme response, pattern recognition receptor response, immune signaling, and modulation of plant hormone levels, among others (Song et al 2020). First, the physical mechanical barrier and chemical defense of plants are critical, including cell wall structural modification and synthesis of antitoxic substances, etc. (Ahuja et al 2012). Meanwhile, plants possess two complex and sophisticated sets of defense systems, PTI and ETI, namely plant immune serration models (Jones and Dangl, 2006). When a plant is attacked by pathogenic bacteria, a receptor on the surface of the cell senses a pathogen related molecular pattern to trigger PTI immune response, including induction of active oxygen burst, hormone signal response, resistance related gene activation and the like; in order to inhibit various effector proteins released by pathogenic bacteria, plants are able to secrete disease-resistant R proteins to further recognize that effector proteins trigger ETI processes, a reaction usually accompanied by cellular programmed necrosis of local tissues, preventing the spread of infection by pathogenic bacteria between cells, ultimately establishing systemic acquired resistance of the whole plant (Zipfel, 2008). Meanwhile, PTI and ETI processes have potentially complex cooperative mechanisms, and receptor and R proteins can act synergistically and activate immune-related pathways in plants (Bruno et al, 2021; yuan et al, 2021). Many disease-resistant related genes have been functionally characterized in cotton, and are widely involved in cell wall modification, secondary metabolic changes, immune receptor recognition and interaction mechanisms with effector proteins, wherein the process of Mitogen Activated Protein Kinase (MAPK) cascade-mediated immune signaling is located in the center of this system, responsible for receiving signals from upstream pattern recognition receptors and amplifying the immune signaling cascade by phosphorylating different substrates, thereby eliciting cellular immune responses.

The mitogen-activated protein kinase (MAPK) cascade is a conserved signaling module in plant evolution that transduces extracellular signals into the nucleus for appropriate cell regulation, mainly by phosphorylation (Meng and Zhang, 2013). The MAPK cascade pathway involves three kinases: mitogen Activated Protein Kinases (MAPKs), MAPK kinases (MKKs) and MAPK kinase kinases (MAPKKKs), which play an important role in both biotic and abiotic stress (Shi et al, 2011). MAPKs are located in the cytoplasm and nucleus and are involved in various cellular processes such as growth, development, and multiple stress responses (Danquah et al, 2015; wang et al, 2015). The evolution of MAPK genes in plants is highly conserved, and MAPK genes from different plants can be identified based on sequence similarity and signature of the TXY motif between kinase subdomains VII and VIII(Hamel et al, 2006). 20 MAPK genes have been identified in Arabidopsis (Ichimura et al, 2002), 17 MAPK genes in rice (Wankhele et al, 2013), 14 MAPK genes in cucumber (Wang et al, 2015), 16 MAPK genes in tomato (Kong et al, 2012), 15 MAPK genes in maize (Kong et al, 2013), 20 MAPK genes in rape (Sun et al, 2014), 16 MAPK genes in brachypodium distachyon (Min et al, 2014), 15 MAPK genes in watermelon (Song et al, 2015), 14 MAPK genes in grapeet al, 2015), 26 MAPK genes in apple (Shizhong et al, 2013), 22 MAPK genes in banana (Asif et al, 2013), 38 MAPK genes in soybean (neukane et al, 2013), 17 MAPK genes in tobacco (Xing et al, 2013).

Plant MAPKs can be divided into two subtypes, the TEY subtype and the plant-specific TDY subtype, which are related to the yeast and animal ERK subfamilies, according to the amino acids in the middle of the TXY motif. Among them, TEY subtypes of plant MAPK can be further divided into three groups: group A, represented by Arabidopsis MPK3 and MPK6, was originally associated with plant immunity and response to abiotic stress, and was later shown to play an important role in plant growth and development (Xu and Zhang et al, 2015; zhang et al, 2018). Tobacco NtSIPK and NtWIPK can be induced by a variety of biotic and abiotic stresses, involved in plant allergic reactions and cell death; rice OsMPK5 is induced by different pathogenic bacteria and environmental stimuli (Soyano et al 2003;Agrawal et al, 2002, xiong et al, 2003). Group B includes arabidopsis MPK4, MPK5, MPK11, MPK12 and MPK13, which are also involved in plant immunity, response to environmental changes and growth development (de Zelicourt et al 2016;Thulasi Devendrakumar et al, 2018). Tobacco ntf6 is also involved in a wide range of signaling processes including various biotic, abiotic stress responses, and cell division activities. Group C comprises Arabidopsis MPK1, MPK2, MPK7 and MPK14, wherein AtMPK1, atMPK2 are mechanically damaged, JA, ABA and H ₂ O ₂ Induction (Ren et al, 2002;Ouaked et al, 2003). Rice OsMPK4 is mechanically damaged, JA, SA, ABA, ET、H ₂ O ₂ And salt stress, sucrose and heavy metal induction expression. The TDY subtype of plant MAPK is also known as group D MAPK, with eight members in arabidopsis, including MPK8, MPK9, MPK15, MPK16, MPK17, MPK18, MPK19 and MPK20. Group D members have an expandable C-terminal region as compared to A, B and group C members. There are studies indicating that arabidopsis MPK9 has autophosphorylation at Thr and Tyr residues of its TDY motif, which does not require upstream MAPKK for activation (Nagy et al 2015). MPK9 may also be involved in ROS-mediated ABA signaling pathways in arabidopsis guard cells along with MPK12 in subgroup B (jammers et al, 2009). In addition, members of group D, osBWMK1, osMPK13, osMPK15, osMPK17, etc., in rice are all subjected to pathogen, SA and ET induced expression (Soyano ET al, 2003;Agrawal ET al, 2002).

The cotton verticillium wilt is a first major disease of cotton, and has wide spread path, and various mediums such as cotton seeds, plant residues, soil, fertilizer water, farm tools and the like can be spread. Wherein the MAPK cascade pathway mediated immune signaling pathway plays a key role in regulating the resistance of cotton to verticillium wilt. Currently, 52 MAPKs, 23 MAPKKs, 166 MAPKKKs are identified in upland cotton and 28 candidate MAPK cascade genes are identified in raymond cotton (Zhang et al, 2014; yin et al, 2021). Wang isolated and cloned a cotton C group MAPK gene GhMPK2 from upland cotton for the first time, and overexpressed GhMPK2 in tobacco, which enhances the resistance of plants to viruses such as tobacco mosaic virus, wilt and the like by regulating plant ROS and ET signal paths (Wang ET al, 2007). Zhang separated subgroup a, subgroup GhMKK1 and subgroup C, ghMKK4 and GhMKK5, respectively, from upland cotton, and increased tolerance of plants to stress and pathogenic bacterial infection after overexpression of GhMKK5 in tobacco (Zhang et al 2012). Shi is separated from upland cotton to two MAPK genes GhMPK7 and GhMPK16, wherein the GhMPK7 participates in regulating the growth and development of plants and SA-mediated broad-spectrum resistance of the plants; overexpression of GhMPK16 in Arabidopsis increased its resistance to Pseudomonas solanacearum (Shi et al, 2010; 2011). MKK members of the MAPK cascade play a dual role in fine control of cotton resistance to verticillium: ghMKK4, ghMKK6 and GhMKK9 positively regulate cotton verticillium wilt resistance, while GhMKK10 negatively regulates cotton verticillium wilt resistance (Meng et al, 2018). Using gene silencing techniques, ghNDR1 and GhMKK2 have proven critical for verticillium resistance in cotton (Gao et al, 2011). The GhMKK4-GhMPK20-GhWRKY40 cascade pathway negatively regulates cotton resistance to wilt (Wang et al, 2018). Overexpression of the scaffold protein GhMORG1 of the GhMKK6-GhMPK4 cascade enhanced cotton resistance to wilt (Wang et al, 2019). The GhMKK2-GhNTF6-GhMYC2 cascade increases resistance to wilt by up-regulating expression of genes involved in flavonoid biosynthesis (Wang et al, 2022). With the intensive research of plant MAPK, more and more MAPK phosphorylating substrates are discovered, which lays a foundation for the deep understanding of the mechanism of MAPK signaling in plant immunity. GhMKK4, ghMKK6 and GhMKK9 positively regulate cotton verticillium wilt resistance, while GhMKK10 negatively regulates cotton verticillium wilt resistance (Meng et al, 2018). Using gene silencing techniques, ghNDR1 and GhMKK2 have proven critical for verticillium resistance in cotton (Gao et al, 2011). Combining MAPK molecular mechanism analysis, further exploring different MAPK cascade pathways of cotton will be the focus of cotton disease-resistant breeding research.

In the research of the invention, the GhMPK9 gene in cotton is cloned and identified, and the GhMPK9 gene is expressed in a large amount under the induction of cotton roots. Through in vivo and in vitro interaction verification of the full-length sequence, the key disease-resistant genes GhRAF39_1 and GhWRKY40a downstream of GhMPK9 are defined, and the GhRAF39_1 is found to play an important role in mediating GhMPK9 phosphorylation GhWRKY40a to regulate cotton verticillium wilt resistance. The research of the invention shows that the over-expression of GhMPK9 improves the resistance of cotton to verticillium. There is little research on GhMPK9 in cotton, and its functional mechanism in plant pathogen resistance has been systematically under investigation. The transgenic plant of GhMPK9 is obtained by the method of gene interference and over-expression, and the important role of GhMPK9 in cotton growth and development and disease resistance is defined. Verticillium wilt is called "cancer" of cotton, and it is particularly critical to discover key genes in plants that prevent their pathogenic agents and to create disease-resistant species by effective genetic engineering means.

Disclosure of Invention

The invention aims to provide an application of cotton GhMPK9 gene in improving disease resistance of target plants or cultivating new germplasm of the target plants with improved disease resistance. The transgenic cotton material proves that the over-expression of GhMPK9 can obviously improve the resistance of plants to verticillium wilt. The gene is used as a target gene, the GhMPK9 gene is excessively expressed in plants by a transgenic isogenetic engineering method, the cotton is cultivated and grown normally, the yield and quality of cotton fiber are not obviously different from those of a control, and the resistance is obviously improved, so that the novel cotton germplasm is applied to production.

Another object of the invention is to provide a method for improving verticillium wilt resistance of cotton.

It is a further object of the present invention to provide a cotton GhMPK9 gene, and to provide the full-length cDNA ORF nucleotide sequence and the encoded amino acid sequence of the gene in the upland cotton A subgroup (GhMPK 9A).

The aim of the invention is achieved by the following technical scheme:

in a first aspect, the invention claims the use of the GhMPK9 gene as shown in SEQ ID NO.1 in improving disease resistance of a target plant or cultivating a novel germplasm of a target plant with improved disease resistance.

In a second aspect, the invention claims the application of a recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing GhMPK9 gene with a nucleotide sequence shown as SEQ ID NO.1 in improving the disease resistance of target plants or cultivating new germplasm of the target plants with improved disease resistance.

Furthermore, the application is specifically that the GhMPK9 gene is taken as a target gene, and the GhMPK9 gene is over-expressed by a genetic engineering method, so that the disease resistance of a target plant is improved or a new germplasm of the target plant with improved disease resistance is cultivated, and the novel germplasm is applied to production.

Further preferably, the disease resistance is verticillium wilt resistance. The target plant is cotton or Arabidopsis thaliana.

In a third aspect, the invention claims a method for improving verticillium wilt resistance of cotton, in which the GhMPK9 gene with a nucleotide sequence shown in SEQ ID NO.1 is overexpressed.

In a fourth aspect, the invention claims a GhMPK9 gene capable of remarkably improving plant disease resistance, wherein the nucleotide sequence of the full-length cDNA ORF of the gene is shown as SEQ ID NO.1 in a genome A subgroup (GhMPK 9A) of upland cotton.

In a fifth aspect, the invention claims a protein encoded by the GhMPK9 gene, the protein having the amino acid sequence shown as SEQ ID NO. 2.

In a sixth aspect, the invention claims a recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the above mentioned GhMPK9 gene.

The research shows that the over-expression of the GhMPK9A gene into cotton can obviously improve the disease resistance of plants without affecting the normal growth and development of the plants. The GhMPK9 gene is used as a target gene to obtain cotton materials with obviously improved disease resistance, and the target gene/locus is introduced into the existing popularization variety by hybridization and backcross transformation with the production-up main-pushed variety to improve the disease resistance of the popularization variety.

The invention has the advantages that:

(1) The sequence structure, expression pattern and function analysis of the GhMPK9 gene in cotton are performed to determine the important role of GhMPK9 in cotton disease resistance.

Through a systematic molecular biological experiment, the reduction of the expression level of GhMPK9 in cotton is proved to reduce the resistance of the cotton to verticillium after verticillium infection, and the inhibition of GhMPK9 gene expression in the cotton influences the reproductive development of plants so that sterile phenotype cannot produce offspring. The overexpression of GhMPK9 gene in cotton has no influence on the growth and development of plants, but after verticillium infection, the immune pathway related to disease resistance is enhanced, and the gene expression is widely related to receptor recognition, hormone signal transduction and disease resistance. The research result reveals the molecular mechanism of GhMPK9 in cotton for regulating and controlling the growth and development of plant reproductive organs and verticillium wilt resistance, and provides a new idea and reference for further researching the research direction of how to accurately balance growth-immunity of plants.

(2) The key disease-resistant genes GhRAF39_1 and GhWRKY40a which are firstly excavated to the downstream of GhMPK9 are found to play an important role in mediating GhMPK9 phosphorylation GhWRKY40a to regulate cotton verticillium wilt resistance.

Up to now, few related studies on the resistance of MAPKs to verticillium have been reported in cotton, and particularly few studies on the detailed molecular mechanism of MAPKs against verticillium have been reported. By integrating the results of a plurality of histology analyses, the key disease-resistant genes GhRAF39_1 and GhWRKY40a which interact with GhMPK9 are mined, and the interaction and regulation relation among the GhMPK9, the GhRAF39_1 and the GhWRKY40a are defined by means of relevant physiological and biochemical experiments. Through the results, theoretical guidance and genetic resources can be provided for molecular design breeding or genetic engineering for improving cotton disease resistance.

Drawings

FIG. 1 characterization of GhMPK9 in cotton

(A) Phylogenetic tree of homologous genes of GhMPK9 in upland cotton and arabidopsis. Gh represents upland cotton G.hirsutum, at represents Arabidopsis Arabidopsis thaliana. Island cotton g. (B) Analysis of expression patterns of GhMPK9 in different tissues and organs of cotton. (C) Expression levels of GhMPK9 in V991 treated RNA-seq samples were normalized by TPM and verified by qRT-PCR. The line graph represents the expression of GhMPK9A/D subgroup in the transcriptome and the bar graph represents the results of qRT-PCR. (D) Alignment of the protein sequences of GhMPK3A/D, ghMPK6A/D, ghMPK A/D, ghMPK13A/D and AtMPK 3/6. (E) subcellular localization of GhMPK9 protein. Green fluorescence represents GFP. Scale = 50 μm.

FIG. 2 creation of GhMPK9 repressed expression transgenic cotton material

(A) The DNA of the GhMPK9-RNAi line was detected by PCR. PC: a positive control; WT: wild type; RNAi GhMPK9 suppresses expression of the transgenic line. (B) Relative Expression Level (REL) of GhMPK9 in wild-type and transgenic plants. (C) The phenotype of stigma, stamen and pistil in wild type and transgenic cotton plants (RNAi-1 and RNAi-2). (D) Phenotype of GhMPK9-RNAi transgenic cotton after artificial pollination. On day 7 of pollination, ovules die off.

FIG. 3 phenotypic analysis after GhMPK9 Gene silencing

(A) Tobacco embrittlement virus (TRV) mediated gene silencing (VIGS) technology silences the phenotype of GhCLA a 1. (B-C) silencing efficiency of GhMPK9 and homologous gene GhMPK13 was verified by qRT-PCR. (D) the plants are inoculated with the pathogenic phenotype of verticillium 15-35 dpi. And (E) counting the leaf disease rate of the plant after verticillium treatment. (F) upper graph: and (3) performing pathogen resuscitating experiments on the stem parts of the plants 13 days after verticillium inoculation. The following figures: and (3) observing the vascular tissue of the stem of the plant after 23 days of verticillium inoculation. FIG. 4 transcriptome sequencing of plants after GhMPK9 Gene silencing

(A) Under water or verticillium treatment, the gene numbers were differentially expressed in roots of TRV: ghMPK9 and TRV:00 plants. The black color in the bar graph indicates the number of up-regulated differential genes and the gray color indicates the number of down-regulated differential genes. (B) GO enrichment analysis of the downregulated expression differential gene between TRV, ghMPK9 and TRV, 00.

FIG. 5 identification of verticillium wilt resistance of GhMPK9 overexpressing transgenic plants

(A) PCR detection of genomic DNA from GhMPK9 transgenic cotton plants. (B) qPCR detection of the transcript level of GhMPK9 in the transgenic line. (C) qPCR detection of the transcript level of GhMPK13 in the transgenic line. (D) Phenotype of transgenic cotton plants 27 days after verticillium inoculation. (E) Leaf disease rate investigation of transgenic cotton and wild type W0 plants. (F) And (5) observing the vascular tissue of the stem of the plant after 13 days of verticillium inoculation. (G) The verticillium is inoculated for 13 days, and the germs on the stem of the plant are recovered for 15 days.

FIG. 6 transcriptome sequencing of GhMPK9 overexpressing transgenic plants

(A-C) differential expression of gene numbers in roots of WT and OE2/OE10 plants under water (A) or verticillium (C) treatment. The black color in the bar graph indicates the number of up-regulated differential genes and the gray color indicates the number of down-regulated differential genes. (B-D) Venn diagram of differential gene showing overlap between up-regulated differential genes between WT and OE2/OE10 under normal (B) and V991 (D) conditions. (E) GO enrichment analysis of overlapping differential genes between WT and OE2/OE 10.

FIG. 7 model of GhMPK9 against verticillium and activating plant immune processes in cotton

When verticillium is invading, ghMPK9 is activated by unknown kinase, ghRAF39_1 and GhWRKY40a downstream of GhMPK9 phosphorylation activation, thereby promoting PR gene expression level and regulating leaf stomata opening. PR expression and leaf moisture loss can inhibit the colonization of fungi, and endow plants with stronger disease resistance.

Detailed Description

The following description of the present invention will be made in further detail by way of example only, and it should not be construed that the scope of the subject matter of the present invention is limited to the following examples, but all techniques which can be implemented in the art based on the foregoing description of the present invention are intended to fall within the present invention.

(one) GhMPK9 full-length sequence acquisition and induced expression pattern analysis and localization analysis

Based on the existing sequence, PCR amplification primers were designed, which were used to clone the full-length ORF of the gene (GhMPK 9) from upland cotton TM-1. The MAPK family members are identified by homologous search from the Lemond cotton genome by taking the amino acid sequences of the MAPKs of Arabidopsis thaliana and rice as references. Intronic/exonic structure of MAPK family genes was predicted by GSDS website. And (3) utilizing MEGA software to cluster MAPK protein kinase to construct a phylogenetic tree. Phylogenetic analysis showed that GhMPK9 and GhMPK13 were highly homologous to Arabidopsis AtMPK3, while GhMPK3 and GhMPC6 were highly homologous to AtMPK6 (FIG. 1A). By aligning the homologous gene sequences of GhMPK9 in cotton and Arabidopsis thaliana, the multiple sequence alignment details that their structural features are very conserved, they are Ser/Thr class protein kinases, all have 11 repeated Ser/Thr sub-domains, there is a conserved activating loop with TEY motif between the VII th and VIII th Ser/Thr sub-domains, in the prior report an ATP-phosphorylating active site that can be double-phosphorylated by MKK, thus activating MAPK (FIG. 1D). To understand the expression pattern of GhMPK9 and homologous genes in tissues and organs, we analyzed the expression levels of roots, stems, leaves, petals, anthers, 0dpa ovule, 10dpa fibers and 20dpa fibers in cotton (TM-1). According to analysis of upland cotton tissue organ expression data, although GhMPK9 and GhMPK13 are highly expressed in flower organs, ghMPK9_A/D and homologous genes GhMPK13_A/D, ghMPK3_A/D and GhMPK6_A/D are higher expressed in roots, stems and leaves, which indicates that the 4 homologous genes can be differentiated functionally (FIG. 1B). According to the analysis of induction expression data of cotton root tissues subjected to verticillium wilt, ghMPK9 is changed in different stress treatment periods, and particularly, the GhMPK9 is obviously up-regulated in the early stage (12 hours) of stress treatment. The induction expression characteristics of the recombinant DNA under the stress of verticillium are verified by qRT-PCR, and are consistent with transcriptome data, and the result shows that the expression level is highest in 12 hours of verticillium infection, which indicates that the accuracy of early analysis is higher, and GhMPK9 can play a role in the verticillium resisting process (figure 1C). The localization of proteins is often related to their function. In order to clearly determine the intracellular localization of GhMPK9, a 35S promoter-initiated GhMPK9-GFP vector was initially predicted by a Softberry website, and the intracellular location of green fluorescence was observed by Agrobacterium transformation of tobacco leaves. After 3 days of Agrobacterium infection, subcellular localization of GhMPK9 was observed under confocal microscopy, and green fluorescent signal localization was observed under confocal microscopy, indicating that GhMPK9 is a nuclear and plasma membrane localization protein (FIG. 1E).

Table 1: primers for amplification

(II) GhMPK9 is a key gene for immunization and reproductive development of cotton plants

In order to deeply explore the action mechanism of GhMPK9 on cotton disease resistance, transgenic cotton materials which interfere with GhMPK9 expression are created. A gene specific fragment with the length of 318bp is connected to a pART27-pKANNIBAL interference vector, and is further transferred into a cotton receptor material W0 by an agrobacterium-mediated genetic transformation method based on hypocotyls. A318 bp specific fragment of the gene of interest was ligated forward to the interfering vector via XhoI and EcoRI and reverse ligated to the interfering vector via HindIII and XbaI. And transferring the connection product into escherichia coli DH5 alpha, and analyzing the bacterial liquid and the sequencing result by PCR to prove that the target fragment is integrated on the vector, so that pART27-pKANNIBAL-35S is successful in constructing the GhMPK9 interference vector. Plasmid DNA of pART27-pKANNIBAL-35S, namely GhMPK9 interference vector, is transformed into competent cells of agrobacterium LBA4404, and the competent cells are successfully transformed into agrobacterium through detection of bacterial liquid PCR, and transgenic cotton with GhMPK9 expression inhibition is obtained through an agrobacterium-mediated cotton hypocotyl genetic transformation method by taking a hypocotyl of cotton material W0 as a receptor. The total DNA of leaves of cotton seedlings was extracted from the obtained 5 clone T0 generation lines by the CTAB method and examined (FIG. 2A). Further verifying the change of the transcription level of the gene in the GhMPK9-RNAi plant, respectively selecting two single plants with good growth conditions for different clones, sampling leaves, extracting RNA, performing reverse transcription to obtain cDNA, and detecting the expression quantity of the GhMPK9 gene by taking the cDNA as a template. The results showed that compared to receptor W0, ghMPK9 gene expression was significantly reduced in 5 positive clones from GhMPK9-RNAi (FIG. 2B). Unfortunately, none of the GhMPK9-RNAi lines was able to set seed. We found that stamen of the GhMPK9-RNAi line developed abnormally and failed to produce viable pollen (FIG. 2C). Thus, we pollinate the W0 receptor's viable pollen to the GhMPK9-RNAi line, but still failed, indicating that pistils also develop abnormally (FIG. 2D). These results indicate that GhMPK9 plays an important role in the development of cotton reproductive organs.

To elucidate the function of GhMPK9, we silenced GhMPK9 in Hai7124 by tobacco embrittlement virus mediated gene silencing (VIGS). Selection of a segment specific for the CDS region of the gene pTRV2 vector for GhMPK9_A (D) (TRV: ghMPK 9) was constructed to silence the endogenous gene, TRV:00 as a negative control. To verify the feasibility of the VIGS system in cotton, leaves and stems of plants after silencing all exhibited a albino phenotype with reference to the CLA1 gene encoding 1-deoxy-D-xylulose 5-phosphate synthase (Gao et al, 2013). Picking single colony of TRV1, TRV2 (negative control), TRV2: CLA1 (positive control) and TRV2 plasmid Agrobacterium GV3101 containing target gene fragment, inoculating 3mL of LB containing corresponding antibiotics (Kan: 100 μg/mL, rif:50 μg/mL), culturing at 28deg.C and 170rpm for 16h, inoculating into 50mL LB liquid medium at 28deg.C and 160rpm for 12h, culturing until bacterial liquid OD ₆₀₀ When the concentration was about 0.5, cells were collected by centrifugation at 1000rpm for 10 minutes, resuspended to a final concentration of 2.0 in an appropriate volume of a resuspension (10 mM MgCl2, 10mM MES and 200. Mu.M acetolyrinone), and the resuspension was allowed to stand at room temperature for 3 hours, and after mixing, used for inoculation. After cotton seedlings grow until two cotyledons are fully unfolded, an agrobacterium inoculation experiment is performed, and uninjected seedlings are used as a control. Cotton leaf injected with TRV/CLA 1 vector after two weeksExhibiting a highly uniform whitening phenotype. Agrobacteria carrying these constructs were injected separately into cotton cotyledons, with uninjected seedlings as controls. When the second leaf of TRV: ghCLA seedlings showed albino phenotype (FIG. 3A), we collected the second leaves of GhMPK 9-silenced seedlings and control material seedlings, respectively, extracted RNAs, and verified the expression level of the gene by a real-time fluorescent quantitative PCR (qPCR) method to verify the silencing efficiency of the target gene (FIGS. 3B-3C). After the target gene silencing is determined, disease resistance identification is carried out in a period from the growth of plants to the two-leaf and one-heart stage under normal greenhouse culture conditions. Using a cultured Verticillium wilt strain V991, 25mL 1X 10 as a control with a disease-sensitive cotton variety upland cotton JM1 ⁷ Is used for treating diseases in a root injury mode. More than 30 plants were planted per material, 3 replicates were set. Compared with TRV:00, the seedling of TRV: ghMPK9 was more likely to develop verticillium wilt (FIG. 3D). The disease condition of the plants was investigated starting 15 days after inoculation, and after 20 days of inoculation, the disease rate of the TRV: ghMPK9 plants was found to be significantly higher than TRV:00, and then the disease rate of the TRV: ghMPK9 plants was always higher than TRV:00 (FIG. 3E). And taking TRV of GhMPK9, TRV of 00 and the same position of the first army cotton stem at 10 days of inoculation, carrying out a germ recovery experiment, and culturing for 3 days, wherein the TRV of GhMPK9 and the first army cotton plant tissue generate a large amount of hyphae in a culture medium, and the TRV of 00 silencing plants do not generate hyphae. After infection by verticillium, plant vascular tissue is blocked, making it brownish black (Fradin and Thomma, 2006). The stem splitting and microscopic observation were carried out on the invasion conditions of the vascular tissues of the stems of different materials at 23 days of pathogen infection, and fungus colonization was hardly observed on the TRV 00-silenced plants (FIG. 3F) due to the blackening of the vascular tissues of the GhMPK9 and army cotton No.1 plants at the position 2cm below the cotyledonary node due to the accumulation of microsclerotia and melanin. The above results indicate that the fungal accumulation in the seedlings of TRV: ghMPK9 is greater than that of the plants of TRV: 00. We performed transcriptome sequencing of root tissue of TRV: ghMPK9 seedlings and compared to TRV:00 under normal and V991 conditions. GhMPK9 had 1034 down-regulated expressed DEGs compared to TRV 00 under normal conditions, and 176 down-regulated expressed DEGs under V991 conditions (FIG. 4A). GO enrichment analysis shows that these down-expressed DEGs are mainly involved in reactions to injury, chitin and fungi,Modulation of plant allergic reactions, jasmonic acid, salicylic acid, abscisic acid and ethylene activated signaling pathways (fig. 4B). These results indicate that inhibition of GhMPK9 expression impairs cotton resistance to verticillium, and that GhMPK9 plays a key role in regulating cotton response to verticillium.

Table 2: primers for detecting transgene target gene on genome and expression level

(III) overexpression of GhMPK9 activates plant immune response to enhance resistance to verticillium wilt

pCAMBIA2301 is a traditional plant binary expression vector, an over-expression vector of a 35S promoter is constructed based on the vector, a full-length fragment of the SEQ NO.1 sequence of the gene is constructed, after the construction is completed, a GhMPK9 over-expression line (OE) driven by the 35S promoter is further generated in cotton through an agrobacterium-mediated genetic transformation system, and is transformed into cotton receptor material W0, so that 10 transgenic clone lines with stable inheritance are respectively obtained. Fertility of the GhMPK9-OE line was not affected compared to the GhMPK9-RNAi line, and there was no significant difference in quality and yield of mature fibers compared to the wild type. Further taking root extraction RNA of different clone lines to detect the transcription level of GhMPK9 gene, and determining that GhMPK9 is over-expressed in plants.

Based on the above results, we selected two stable over-expressed transgenic lines, OE2 and OE10, in which expression of GhMPK9 was significantly increased, while its homolog, ghMPK13, was unchanged to further elucidate the response function of GhMPK9 to verticillium (fig. 5A-5C). Inoculating pathogenic bacteria spore suspension when cotton seedling grows to "two leaves one heart" stage, cutting the bottom of the nutrition pot by root injury inoculation method to achieve root injury, and regulating verticillium wilt bacteria spore suspension concentration to 10 ⁷ Individual spores per milliliter, at each newThe disposable paper cup is evenly filled with 25mL, the seedling with the cup bottom removed is placed in a new paper cup containing bacteria liquid, and the light growth is carried out for 16 hours and the dark culture is carried out for 8 hours (25 ℃/23 ℃). Subsequent observations found that WT showed significant cotyledon wilting and partial yellowing of true leaves from 13 days to 27 days (dpi) after inoculation, with the true leaves increasingly yellowing off over time. However, the GhMPK9 over-expressed transgenic line exhibited lighter disease symptoms compared to the wild type (fig. 5D). From 13 to 34dpi, the foliar rate of GhMPK9-OE plants was significantly lower than that of wild-type seedlings (FIG. 5E). In addition, stems of wild type and over-expressed plants were cut into stem segments of about 5mm at 10dpi infection with V991 and cultured on PDA solid medium for pathogen recovery experiments. After 3-5 days, the fungal recovery in WT was significantly higher than OE2 and OE10 (FIG. 5G). At 13dpi, V991 mycelia accumulated far more in WT than in the OE line (FIG. 5F). In conclusion, overexpression of GhMPK9 increased resistance to verticillium in cotton.

Further, the molecular mechanism for improving cotton verticillium resistance by analyzing the overexpression of GhMPK9 through transcriptome sequencing. Root tissues of OE2, OE10 and W0 were taken 3 days after water and verticillium treatment, respectively, for RNA-seq sequencing analysis. The original reads of the transcriptome sequencing machine were first quality controlled by the Cutadapt software, then the clean reads of all sequencing libraries were aligned to the upland cotton reference genome by the HISAT2 software, PCR replicates were removed using Picard and low quality reads were filtered out using Samtools, filter reads for all cotton annotation genes were counted using HTSeq, and the count files were used for differential expression analysis using the R software packages DESeq2 and edge. The assembly and quantification of transcripts was performed by StringTie, and gene expression levels were normalized by means of Transcripts Per Kilobase of exon model per Million mapped reads (TPM) for subsequent analysis. We compared the differentially expressed genes between GhMPK9 over-expressed material and wild-type material under normal and V991 inoculation conditions. Under normal conditions there were 1624 up-regulated Differentially Expressed Genes (DEG) in OE2, 3761 up-regulated DEGs in OE10 and 1458 overlapping DEGs compared to the wild-type (FIGS. 6A-6B). Under V991 inoculation conditions, we identified 1709 upregulated DEGs in OE2 and 613 upregulated DEGs in OE10, 255 overlapping DEGs (fig. 6C-6D). GO enrichment analysis showed that under both conditions these overlapping genes were enriched in wound response, osmotic signaling MAPK cascade, plant protection element biosynthesis process, initiation of cellular stress response, detection of biostimulation, regulation of stomatal complex development and pattern (fig. 6E). Overall, overexpression of GhMPK9 activates the immune response of the plant and regulates stomatal movement, thereby enhancing resistance upon verticillium attack.

Table 3: primers for amplification

(IV) model of GhMPK9 in cotton against verticillium and activating plant immune Process

Taken together, we propose that GhMPK9 can be used as a positive regulator to improve the resistance of cotton to verticillium. We demonstrate that GhMPK9, by recognizing pathogenic bacterial infection, when infected by the bacterial, ghMPK9 is activated by unknown kinase, and GhMPK9 phosphorylates downstream substrates, while at the same time activating a more intense immune response to improve plant disease resistance. The inhibition of fungal colonization by activating downstream disease resistance gene expression and regulating leaf moisture loss increases cotton resistance to pathogens (FIG. 7). The method is hopeful to complete disease resistance improvement of the popularization variety by over-expressing GhMPK9 transgenic cotton material, crossing and backcrossing with the main-pushing variety in the current production, introducing the target gene into the current upland cotton popularization variety, and creating new upland cotton materials and new lines with high disease resistance, high yield and high quality by combining with screening of fiber quality and yield traits.

Claims

1. The GhMPK9 gene shown in SEQ ID NO.1 is applied to improving the disease resistance of target plants or cultivating new germplasm of the target plants with improved disease resistance.

2. The application of the recombinant vector, the expression cassette, the transgenic cell line or the recombinant bacteria containing the GhMPK9 gene with the nucleotide sequence shown as SEQ ID NO.1 in improving the disease resistance of target plants or cultivating new germplasm of the target plants with improved disease resistance.

3. Use according to claim 1 or 2, characterized in that: the GhMPK9 gene is used as a target gene, and the GhMPK9 gene is over-expressed by a genetic engineering method, so that the disease resistance of a target plant is improved or a new germplasm of the target plant with improved disease resistance is cultivated, and the GhMPK9 gene is applied to production.

4. A use according to claim 1, 2 or 3, characterized in that: the disease resistance is verticillium wilt resistance.

5. A use according to claim 1, 2 or 3, characterized in that: the target plant is cotton or Arabidopsis thaliana.

6. A method for improving verticillium wilt resistance of cotton is characterized by comprising the following steps: the GhMPK9 gene with the nucleotide sequence shown as SEQ ID NO.1 is overexpressed in cotton.

7. A GhMPK9 gene for improving disease resistance of plant has the nucleotide sequence of full-length cDNA ORF shown in SEQ ID NO. 1.

8. A protein encoded by the GhMPK9 gene as claimed in claim 7, which has an amino acid sequence as shown in SEQ ID NO. 2.

9. A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium comprising the GhMPK9 gene of claim 7.