CN109402147B - Cotton verticillium wilt resistant gene GbCYP86A1-1 and application thereof - Google Patents

Abstract

The invention discloses a cotton verticillium wilt resistant gene GbCYP86A1-1 and application thereof, belonging to the field of biotechnology application. The invention relates to a GbCYP86A1-1 gene which codes a fatty acid omega-hydroxylase. The invention provides a genome A subgroup and a genome D subgroup of GbCYP86A1-1 in an alloptetraploid sea island cotton H7124, a full-length ORF nucleotide sequence (without an intron in the gene) and an amino acid sequence. CYP86A1-1 gene in cotton is specifically expressed in root and is induced by verticillium dahliae to be obviously up-regulated. Compared with the susceptible upland cotton variety army cotton No.1, the gene has obviously enhanced expression and induced expression level in the tissues of the disease-resistant sea island cotton variety H7124. Through the identification of verticillium wilt resistance, the verticillium wilt resistance of cotton can be obviously reduced by silencing the gene in the sea island cotton H7124 based on the VIGS technology, and the disease resistance of transgenic arabidopsis can be obviously improved by over-expressing the gene in arabidopsis.

Description

Cotton verticillium wilt resistant gene GbCYP86A1-1 and application thereof

Technical Field

The invention belongs to the field of biotechnology application, and relates to a cotton GbCYP86A1-1 gene and application thereof, wherein the gene codes a fatty acid omega-hydroxylase. Through systematic analysis of CYP86 subfamily members in cytochrome P450 gene families in cotton, a target gene capable of remarkably improving verticillium wilt resistance of cotton and arabidopsis thaliana is excavated. CYP86A1-1 gene in cotton is specifically expressed in root and is induced by verticillium dahliae to be obviously up-regulated. Compared with the susceptible upland cotton variety army cotton No.1, the gene has obviously enhanced expression and induced expression level in the tissues of the disease-resistant sea island cotton variety H7124. The genome sequence, the full-length ORF sequence and the coded amino acid sequence of the gene are obtained in the sea island cotton H7124 by utilizing the PCR technology. The disease resistance and function of the gene are researched by utilizing biotechnology. Through the identification of verticillium wilt resistance, the verticillium wilt resistance of cotton can be obviously reduced by silencing the gene in the sea island cotton H7124 based on the VIGS technology, and the disease resistance of transgenic arabidopsis can be obviously improved by over-expressing the gene in arabidopsis.

Background

During the long-term interaction and co-evolution of plants with pathogens, plants exhibit a specific ability to defend against various pathogens, which is pathologically referred to as disease resistance. Disease resistance has long been described and classified from different perspectives on the phenomena observed in each. The resistance of plants to disease following pathogen invasion is manifested primarily by two pathways. On the one hand, some tissues of the plant are changed in structure or the synthesized chemical components have disease resistance, and the former mainly has cell wall suppository, cutin, wax and other special structures, which are called structure resistance. On the other hand, after the pathogenic bacteria break through the inherent defense barrier of the plant, the plant can express a series of physiological and biochemical disease-resistant reactions to the pathogenic bacteria to deal with the invasion of the pathogenic bacteria, namely, physiological and biochemical resistance (conath et al, 2002).

The resistance of the tissue structure of plants can be divided into two categories: intrinsic structural resistance and induced structural resistance. The research finds that the cotton varieties with different resistances have differences in tissue structures, particularly the differences among vascular bundles. The number of cells in unit area of the disease-resistant variety is more than one time than that of the disease-sensitive variety, the disease-resistant variety has solid xylem and smaller intercellular gap and thicker cell wall compared with the disease-sensitive variety, the arrangement of duct cells is tighter, the deposition amount of callose is obviously increased, the bacterial count of pathogenic bacteria is obviously lower than that of the disease-sensitive variety, the structural change improves the mechanical disease resistance of cotton, reduces the success rate of invasion of verticillium wilt bacteria, and ensures that the bacteria are difficult to expand in plants (Zhang et al, 2013; Mace et al, 2010). The induced structural resistance means that after being induced by pathogenic bacteria, plants induce a series of metabolic changes in the plants through a complex molecular regulation mechanism, and morphological structure changes appear on a cellular level to resist invasion and expansion of external pathogenic bacteria, so that the plants obtain resistance finally. The induced resistance is mainly manifested by cell wall and tissue thrombosis, lignification, ductal parenchymal hyperplasia, and the formation of colloids or infiltrates, among others (Bell et al, 1967). Numerous studies have reported that, when a pathogen infects a plant, a large amount of gum material is formed, thereby hindering the invasion of the pathogen, and in diseased plants, it can be observed that an invaded body or gum is present in the vessel, the vein parenchyma cells are distorted into irregular shapes, the vessel wall is thickened, and a large amount of polysaccharide material is present in the root (Daayf et al, 1997).

The physiological metabolism of plants is changed under the induction of pathogens, and various physiological and biochemical changes occur in plants, such as the accumulation of plant defensins, the burst of active oxygen, the change of plant defense enzyme systems and the expression of disease course related protein genes. When pathogenic bacteria infect plants, the inducer enables host plants to generate and accumulate a class of low molecular weight antibacterial secondary metabolites called plant defensins, and the generation and accumulation of the plant defensins are one of important factors for preventing the plants from being damaged by some pathogenic bacteria and are also chemical bases for resisting diseases of the plants. Plants produce active oxygen, which is the earliest disease resistance response. The host plant generates a large amount of active oxygen in a short time under the action of the inducer, and the active oxygen substances have direct antibacterial effect and can kill pathogenic bacteria. However, high concentrations of reactive oxygen species can damage plant cells and the concentration of reactive oxygen species needs to be appropriately regulated by protective enzymes in the plant. Meanwhile, the active oxygen also improves the capability of resisting the invasion of pathogenic bacteria by enhancing the lignification degree of the cell wall of host plant tissues, the oxidative crosslinking of hydroxyproline glycoprotein, regulating the activity of protective enzyme and the like (Alvarez et al, 2008). When cotton is infected by verticillium wilt, antioxidant and anti-hydrolytic enzymes such as peroxidase (ROD), superoxide dismutase (SOD), glutathione-S-transferase (GSTs), Phenylalanine Ammonia Lyase (PAL) and polygalacturonase inhibitory protein (PGIP) can be timely regulated and expressed, so that the degradation of plant cell walls by verticillium wilt is slowed down and inhibited (James and Dubery et al, 2001). The pathogenesis-related Proteins (PRs) are induced to express by the stress of pathogens, and are proteins which are generated by plants in pathological environments and can directly attack pathogenic bacteria. The induced expression of the disease-course related protein is considered to be one of the biochemical indicators of disease resistance response of some plants.

Complex and perfect disease-resistant signal transmission networks exist in plants. Although the research is not thorough, scientists have summarized the results of the innate immune system of plants and proposed a classical zipper model, and the sawing action of plants against pathogens also represents the importance of the innate immune system to reject a large number of pathogens (Jones and Dangl et al, 2006). Some cell membrane surface receptors play an important role in the transmission of signals. There are studies that show that ISO1 receptor-like protein kinases can interact with FLS2 and ERF in a ligand-like manner, modulating pathogen-associated molecular patterns (PAMPs), triggering immunity in plants (Chen et al, 2014). RLP30, SOBIRl, BAKl contribute to defense against the spoilage fungus, RLP30-2, SOBIRl1-12, BAKl-5 mutants are susceptible to Rhizoctonia sojae and Botrytis cinerea (Zhang et al, 2013). The transcription factor families related to disease-resistant signal transduction in plants mainly comprise WRKY families, bZIP families, ERF transcription factors and MYB transcription factors. WRKY transcription factors may positively or negatively regulate plant disease resistance responses, such as: OsWRKY45 and OsWRKY23 genes in rice respond to the induction of rice blast bacteria, and after overexpression in arabidopsis thaliana, the resistance to pseudomonas is enhanced (Qiu et al, 2009; jin et al, 2009), so that the rice blast bacteria has a positive regulation effect in plant disease resistance; plants were more susceptible to disease after overexpression of the AtWRKY38 and WRKY62 genes in Arabidopsis, while single mutants of either WRKY38 or WRKY62 or WRKY38/WRKY62 double deletion mutants all showed increased resistance to Pseudomonas syringae (Kim et al, 2008). Research in recent years has shown that WRKY transcription factors play a role in the regulation of expression of disease-course-associated protein genes (PRs). The ERF transcription factor is also called disease course related transcription factor, is widely involved in the expression regulation of disease-resistant genes, and the coded protein can be specifically combined with a GCC-box original in a PR gene promoter to regulate the expression of disease course related protein genes, thereby playing an important regulation role in disease-resistant reaction (Bttner and Singh et al, 1997; Dubouzet et al, 2003).

An immune mechanism is induced by signal network transmission in plants, and one typical symptom is that the plants generate Hypersensitivity (HR) reaction to cause necrosis of cells and tissues at infected parts, thereby preventing the spread of pathogenic bacteria. Subsequently, the plants are resistant to pathogens within a certain period of time, which is called Systemic Acquired Resistance (SAR), and simultaneously induce a series of timely and effective defense responses to the plants, mainly manifested by accumulation of secondary metabolites, burst of active oxygen, modification of cell walls, and induced expression of PR (pathogenesis-related proteins), etc., and the occurrence of local disease-resistant responses further activates signal molecules to stimulate immune responses of the whole plants (Feys and Parker et al, 2000; Koornneef and Pietes et al, 2008). Studies have shown that lipid molecules may cause SAR as a signal for class movement. The DIR1 mutant obtained a local anti-disease response following pathogen treatment, and DIR1 had sequence similarity to Lipid Transporters (LTPs) and probably functioned in signaling (Maldonado et al, 2008). Meanwhile, active oxygen plays an important role in plant defense reaction and SAR, and the expression of PR protein is a mark of SAR.

Verticillium dahlia (Verticillium dahlia) is a soil-borne fungal pathogen that infects over 200 dicotyledonous plants, including cotton, tomato, cucumber, pepper, eggplant and potato major commercial crops, causing significant economic losses each year (Fradin and Thomma et al, 2010; Inderitzin and Subbarao et al, 2014). In nature, the life cycle of verticillium wilt is signaled by the roots of the plant in the form of microsclerotia and germinates, the germinating hyphae penetrating the root surface of the plant and then colonizing in the xylem vessels (Vallad and subcarao et al, 2008; Prieto et al, 2010; Zhao et al, 2014), once colonized, the fungus will grow up the vascular tissue and constantly produce conidia and microsclerotia, which will prevent the transport of water and ultimately lead to retarded, wilted or fallen leaves of the plant (Klimes et al, 2015). Since microsclerotia can survive in plant residues and in soil for long periods of time, while the number of plant resistance genes is very limited (Klosterman et al, 2015), verticillium wilt remains difficult to control, especially for some commercial crops, such as cotton (Bejaranoalcazar et al, 1995; Wang et al, 2004). The first line of defense of plants against the invasion of pathogenic bacteria is the epidermal cell wall of the roots of plants, which is the most important layer of defense barrier of plants against pathogenic bacteria, and the most of pathogenic bacteria are prevented. Since verticillium wilt is a disease infected by roots, the resistance of tissue structure induced by pathogenic bacteria plays an important role, and the change of tissue structure can activate the whole immune regulation of plants.

Cork is a complex chemical bio-polyester, mainly containing some fatty substances, and usually deposited on the cell wall of specific tissues, such as root cortex, outer cortex, periderm, seed coat and other marginal tissues, to form cork layer. The cork layer serves as a protective barrier, plays an important role in controlling radial moisture and nutrient element transportation of the root system, and can effectively resist invasion of pathogenic bacteria (Franke and Schreiber et al, 2007). Potato tubers suffering from wounds induce a cork around the skin, increasing resistance to pathogenic bacteria (Lulai and Corsini et al, 1998; Yossi et al, 2011). In addition, wood bolting also increases the resistance of soybeans to phytophthora, thereby avoiding root and stem rot (ranathange et al, 2008). Inoculation with phytophthora increases the suberization of the root-coat and the endothelial layers of the soybean. Further studies have also found that hyphal growth is delayed in varieties with high suberin content (Thomas et al, 2007). Invasion of pathogenic bacteria has also been found to induce a wood-bolting response in other plants, such as Arabidopsis, wheat and tomato (Southeron and Deverall et al, 1990; Franke et al, 2005; Botela et al, 2005). So that the cork can block the invasion of germs by enhancing the physical barrier of root or periderm cell wall, but the role of key genes for controlling cork synthesis in plant biotic stress resistance needs to be deeply studied.

At present, the composition of the suberized cell wall is preliminarily known, but the structure of the suberized polymer is still poorly understood, and the chemical composition thereof needs to be further identified. Omega-hydroxy fatty acids and alpha, omega-dicarboxy fatty acids of C16-C18 (alpha, omega-diacids for short) are the main monomers constituting suberin in plant roots, and are derivatives of fatty acids formed under catalysis of cytochrome P450 omega-hydroxylase (Werckreichhart and feyeresen et al, 2000; Nawrath et al, 2002; Pollard et al, 2008). In the cell pigmentThe CYP86 subfamily of the P450 monooxygenase family is primarily involved in the hydroxylation of the omega site of fatty acyl-COA to form omega-hydroxy fatty acids, while a portion is oxidized to alpha, omega-diacids (Agrawal and Kolattukudy et al, 1978; Kurdyukov et al, 2006; Molina et al); the hydroxylated fatty acids are finally transported to the cell wall through a series of polymerization reactions, and some reductases and transferases are found to participate in the complex process; while the monomers eventually need to be transported across the membrane to the cell wall. The mechanism of transmembrane transport of suberin monomers has only recently been studied, with the ATP-binding cassette (ABC) transporters and Lipid Transfer Proteins (LTPs) involved in transmembrane transport of cutin and wax to the cell wall being the most critical candidate proteins (Pighin et al, 2004; Choi et al, 2011; Deeken et al, 2016). The CYP86 subfamily plays a crucial role in the biosynthesis of suberin. CYP86A1 was first cloned in Arabidopsis, and expression in yeast cells was found to be involved in hydroxylation of short-chain fatty acids (Benveniste et al, 1998). The root systems C16 and C18 of arabidopsis mutant CYP86A1 plants have obviously reduced suberin monomer content, the total aliphatic suberin monomer content is reduced by 60%, and the RT-PCR, GUS staining and cell localization prove that the CYP86A1 is positioned in the root endoplasmic reticulum, which also reflects that the synthesis of the suberin monomer is carried out on the endoplasmic reticulum

et al, 2008). After the homologous gene CYP86A33 of CYP86A1 in the potato is silenced, the periderm suberin content is obviously reduced (Serra et al, 2009). CYP86A2, CYP86A4 and CYP86A8 in Arabidopsis are three genes which are relatively close in homology relationship, play a role in the biosynthesis of extracellular lipids, and are also involved in the hydroxylation of short-chain fatty acids, but most of the research results at present indicate that the three genes are involved in the formation of cell wall cutin, and related fatty acids in which CYP86A2 is involved in the synthesis may inhibit the expression of type III gene in pathogenic bacteria (Xiao et al, 2004; Libeissonet al, 2009; Wellesen et al, 2001). CYP86B1 and CYP86A1 belong to different subclasses of a subclass and are used as long-chain fatty acidsHydroxylase, involved in the hydroxylation of long chain fatty acids from C22 to C24 (Compagnon et al, 2009). The roots of the cyp86B1 mutant in arabidopsis and the C22 and C24 omega-hydroxy acids and alpha, omega-diacids in the seed coat were almost completely absent, but the suberin monomers C22 and C24 fatty acids were increased in accumulation (Molina et al, 2009).

At present, few studies on biosynthesis of suberin in cotton are related, and no reports on CYP86 family genes exist. Cotton, one of the world's important commercial crops, is an important source of cotton fiber as well as cottonseed oil. However, the cotton is very serious in verticillium wilt, and because the pathogenic fungi have the characteristics of wide distribution range, serious harm, wide host range, high propagation speed, long survival time in soil and the like, the cotton is difficult to control, the yield and the quality of the cotton are seriously influenced, and the cotton is called as 'cancer' of the cotton. Therefore, the success rate of preventing and treating verticillium wilt is critical. At present, for verticillium wilt pathogen mechanism, a dispute 32429is introduced, the mechanism of interaction between cotton and verticillium wilt pathogen is still unclear, the cell wall tissues of plant roots are important barriers for preventing pathogen invasion, and research on synthesis related genes of some cell wall biopolyesters in cotton plays an important guiding role in preventing and controlling verticillium wilt pathogen.

Disclosure of Invention

The invention aims to provide application of a cotton GbCYP86A1-1 gene in improving disease resistance of cotton and breeding new cotton germplasm. The gene transient silencing technology and transgenic arabidopsis thaliana prove that the gene relates to the resistance of cotton and arabidopsis thaliana to verticillium wilt. The gene is used as a target gene, and the GbCYP86A1-1 gene is overexpressed by gene engineering methods such as transgenosis and the like, so that a novel cotton germplasm which has good growth and development, normal cotton fiber yield and quality and remarkably improved resistance is cultivated and applied to production.

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

The invention also aims to provide a cotton GbCYP86A1-1 gene and a genome and full-length cDNA ORF nucleotide sequence of the gene in the H7124A subgroup (GbCYP86A1-1A) and the D subgroup (GbCYP86A1-1D) of the gossypium barbadense. The gene has the amino acid sequence coded in subgroup A and subgroup D in sea island cotton.

The purpose of the invention is realized by the following technical scheme:

the GbCYP86A1-1 gene shown in SEQ ID NO.1 or SEQ ID NO.2 is applied to improving the verticillium wilt resistance of cotton and model plant Arabidopsis thaliana and cultivating new cotton germplasm.

The above application, which consists in: the disease resistance of arabidopsis is improved by using the overexpression of the GbCYP86A1-1 gene in arabidopsis. The GbCYP86A1-1 gene is used as a target gene, and the GbCYP86A1-1 gene is overexpressed in cotton through gene engineering methods such as an overexpression technology and the like, so that a novel cotton germplasm which is normal in growth and development and remarkably improved in verticillium wilt resistance is cultivated and applied to production.

A method for improving verticillium wilt resistance of cotton comprises the step of over-expressing GbCYP86A1-1 gene with a nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.2 in the cotton.

A gene GbCYP86A1-1 capable of remarkably improving disease resistance of cotton and arabidopsis thaliana, wherein the gene is any one of (1) to (3):

(1) the gene is in a genome A subgroup (GbCYP86A1-1A) of sea island cotton H7124, and the nucleotide sequences of the genome and the full-length cDNA ORF of the gene are shown as SEQ ID NO. 1;

(2) the gene is in the genome D subgroup (GbCYP86A1-1D) of the sea island cotton H7124, and the nucleotide sequences of the genome and the full-length cDNA ORF are shown as SEQ ID NO. 2.

(3) Nucleotide sequences which are derived from other cotton seeds, have more than 98 percent of homology with the nucleotide sequences of the genes in (1) or (2) and have the same or similar functions.

The protein coded by the gene GbCYP86A1-1, such as the gene GbCYP86A1-1A in (1), has an amino acid sequence shown as SEQ ID NO. 3; the gene GbCYP86A1-1D in (2), wherein the protein coded by the gene has an amino acid sequence shown in SEQ ID NO. 4.

A recombinant vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the GbCYP86A1-1 gene.

The recombinant vector, the expression cassette, the transgenic cell line or the recombinant strain are applied to improving the disease resistance of cotton and cultivating new cotton germplasm.

The disease resistance is verticillium wilt disease resistance. The new germplasm is a new cotton germplasm with significantly improved verticillium wilt resistance.

The invention has the advantages that:

(1) the structural and functional analysis of the system is carried out on CYP86 subfamily genes in cotton for the first time, and the important function of the cork synthesis related gene GbCYP86A1-1 in cotton verticillium wilt resistance is identified.

To date, there have been no reports of studies relating to members of the fatty acid ω -hydroxylase CYP86 gene family in cotton. We have performed detailed analysis of all CYP86 subfamily members in cotton with reference to the genome of cotton, and systematically analyzed the family genes by structure, evolution, and expression. CYP86A in cotton has three homologous genes which are distributed on different chromosomes respectively, but the functions are differentiated, and molecular biology experiments find that the important role of GbCYP86A1-1 in the formation of cell wall fatty acid biopolyester and the induction of verticillium wilt immune resistance is played, which is also the first time that CYP86 subfamily members in cytochrome P450 superfamily in cotton are researched by a systematic method, and provides reference and new genes for the research of root biopolyester in cotton and the breeding of cotton disease-resistant molecules.

(2) The transgenic arabidopsis thaliana overexpressed by GbCYP86A1-1 is an excellent material for researching the change of disease resistance caused by genes and the affected disease-resistant signal path and molecular mechanism, and the transgenic arabidopsis thaliana plant disease resistance can be enhanced by overexpression of GbCYP86A 1-1.

The disease resistance identification of transgenic arabidopsis shows that the overexpression of GbCYP86A1-1 can enhance the resistance of arabidopsis to verticillium wilt, compared with wild type materials, the roots of transgenic plants are more difficult to invade by pathogenic bacteria, the biomass of verticillium wilt bacteria in each tissue is less, and the disease index is lower. In addition, in transgenic plants, not only are certain secondary metabolic process changes related to the synthesis of the biological polyester and changes of ATP-binding cassette (ABC) transport proteins and Lipid Transfer Proteins (LTPs) involved in the transmembrane transport of lipid to cell walls found, but also the enhancement of disease resistance related immune pathways are found, wherein the changes of receptor-like protein kinase and receptor-like protein, ERF disease course related transcription factor and WRKY transcription factor and a large number of PR disease course related proteins are included, and the expression of the genes shows remarkable increase. From the results, the transgenic plants can be found to enhance the physical structure resistance of roots and enhance some induced disease-resistant pathways excited by signal molecules, so that new ideas and references are provided for the research direction of some pathway genes participating in secondary metabolism, and meanwhile, the discovery of GbCYP86A1-1 gene regulation disease resistance lays a foundation for further mining cotton disease-resistant genes and breeding disease-resistant varieties.

Drawings

FIG. 1 systematic analysis of diploid Ramond cotton GrCYP86 gene family members

a: evolutionary analysis of members of the GrCYP86 gene family. b: homology comparison of members of the GrCYP86 gene family. c: structural analysis of the gene of the GrCYP86 gene family member. d: GrCYP86 gene family member domain.

FIG. 2 expression analysis of GhYP 86 Gene family members

a: spatial expression pattern analysis of the GhYP 86 gene family members in different tissues and organs. b: three genes GhYP 86A1-1, GhYP 86A1-2 and GhYP 86A1-3 specifically expressed in roots and GhYP 86B1-1 preferentially expressed in roots are expressed and changed at different times under the induction of verticillium wilt. Disease-resistant material H7124 and susceptible material army cotton No.1 are used as comparison materials.

FIG. 3 validation of VIGS Gene silencing System

a: sea island cotton which silences CLA1 gene shows a phenotype of leaf whitening. b: the phenotype of the disease-resistant material H7124 and the disease-sensitive material army cotton No.1 verticillium wilt bacteria V991 treatment and water treatment is compared. c: VIGS separately silences the effects of GbCYP86A1-1, GbCYP86A1-2 and GbCYP86A1-3 on the expression of two other homologous genes.

FIG. 4 silencing GbCYP86A1-1, GbCYP86A1-2, GbCYP86A1-3 plant susceptibility improvement in sea island cotton

A: and carrying out quantitative PCR verification on the gene expression level after separately silencing GbCYP86A1-1, GbCYP86A1-2 and GbCYP86A1-3 and jointly silencing the three genes in the sea island cotton. B: after VIGS silencing gene, verticillium wilt pathogens are inoculated to the susceptible phenotype at different time. C: and (4) investigating the disease grade of the plants after silencing different genes. D: the cotyledon node is upward 1cm after 15 days of inoculation, and the stem of the plant has a split-stem phenotype.

FIG. 5 phylogenetic analysis of chromosomal location, subcellular localization and different species isogenic Gene location of three isogenic genes GbCYP86A1

a: the location of the GbCYP86 family homologous gene on the chromosome of cotton Ramond. b: phylogenetic analysis and homology comparison of CYP86A1 genes from different species. c: subcellular localization of three genes of GbCYP86a 1.

FIG. 6 identification of disease resistance of transgenic Arabidopsis thaliana with three genes GbCYP86A

a: molecular detection of transgenic Arabidopsis thaliana. b: phenotype of transgenic arabidopsis 2 weeks after inoculation with the different vectors. c: statistics of disease grades of different vectors and analysis of disease grade indexes. d: the relative content of verticillium wilt in roots, stems and leaves after different vectors are inoculated for 10 days. e: and (3) carrying out germ recovery analysis on stem sections at 1cm above the lotus throne leaves of different carriers after culturing for 3 days in a potato culture medium.

FIG. 7 GbCYP86A three genes transgenic Arabidopsis plant phenotypic characteristics.

Compared with the growth and development of the wild arabidopsis thaliana, the transgenic arabidopsis thaliana which grows in the soil pot for 4 weeks has no obvious change in phenotype.

FIG. 8 microscopic observation of three genes of GbCYP86A transgenic Arabidopsis thaliana seedling roots.

a: root-length phenotype. b: root cell length. c: root hair phenotype. d: and (5) counting the root length. e: and (5) counting the length of the root cells. f: and (5) counting the length of the root hair.

FIG. 9 is the enrichment analysis of GbCYP86A1-1 transgenic Arabidopsis root transcriptome GO, the tissue section microscopic observation of the root of GbCYP86A three-gene transgenic Arabidopsis at the mature period, and the detection of the relative content of C16-C18 fatty acids.

a: comparing the difference of root transcriptome of GbCYP86A1-1 transgenic Arabidopsis with that of wild Arabidopsis, and the biological process of GO enrichment. b: taking a sample of the main root of the arabidopsis thaliana in the mature period, wherein the sample is 2cm away from the ground base part, and carrying out tissue sectioning. c: the samples were prepared into ultrathin sections, and were cut at the middle to a thickness of 2 μm for lipid staining. d: the relative content of fatty acids in the roots was determined.

FIG. 10 GbCYP86A1-1 transgenic Arabidopsis thaliana and control root transcriptome sequencing identified differential expression gene

On the basis of statistical difference, differential expression genes with the FDR less than 0.05 are extracted, wherein the genes with more than 1.5-fold difference are roughly divided into two types and respectively participate in a secondary metabolic process and a disease-resistant immune process.

FIG. 11 GbCYP86A1-1 transgenic Arabidopsis thaliana verticillium wilt germ 3 days after treatment root transcriptome sequencing analysis and free-hand slicing of roots

a: GbCYP86A1-1 transgenic Arabidopsis and wild Arabidopsis are subjected to verticillium wilt treatment and water treatment respectively, and then the number of different genes is compared, and GO enrichment is performed respectively. b: and (4) slicing the roots by hands after the verticillium wilt is infected for 3d, and observing the invasion condition of pathogenic bacteria.

FIG. 12 quantitative PCR validation of transcriptome analysis results

a: randomly selecting 30 genes with the multiple difference of more than 1.5 times of the transcriptome sequencing result after water treatment to perform quantitative PCR verification, drawing a scatter diagram to calculate R²The value was checked for fitness. The abscissa is the difference multiple of GbCYP86A1-1/WTBase values in the transcriptome sequencing result, and the result is taken as log 2; the ordinate represents the quantitative PCR result 2^-△CtThe result is taken as log 2. b: the same 30 genes are verified by quantitative PCR after verticillium wilt bacteria V991 treatment.

FIG. 13 quantitative PCR detection of genes involved in secondary metabolic process and disease-resistant immune process in GbCYP86A1-1 transgenic Arabidopsis and wild Arabidopsis

a: and participating in quantitative PCR verification of secondary metabolism related genes. b: quantitative PCR verification of receptor-like protein kinases (RLKs) and receptor-like proteins (RLPs). c: quantitative PCR validation of related transcription factors (ERFs, WRKYs). d: quantitative PCR verification of disease course associated protein genes (PRs).

FIG. 14 Effect of silencing GbCYP86A1-1 Gene on expression of Process-related protein Gene in Cotton

a: the expression changes of 8 disease course related protein genes in the sea island cotton at different times under the induction of verticillium wilt bacteria. b: compared with a control, the disease course related protein genes PR1 and PR5 are greatly and obviously down-regulated to express after the GbCYP86A1-1 gene is silenced, and PR2, PR4 and PR16 are obviously and down-regulated to express.

FIG. 15 GbCYP86A1-1 gene affecting the resistance of plant cell wall structure and physiological and biochemical resistance

The GbCYP86A1-1 gene can promote the cell wall to be embolized after being induced by verticillium dahliae, so that the plant can generate induced structure resistance; the change of lipid and the modification of cell wall can affect physiological and biochemical resistance and activate immune response in cells.

Detailed Description

The invention utilizes a bioinformatics method to identify 10 CYP86 family genes from the genome of the released diploid Ramond cotton, and further explores homologous genes from the sequenced Gossypium hirsutum TM-1 and Gossypium barbadense 3-79, and the genes are classified into A, B, C by systematic cluster analysis. Tissue organ expression analysis shows that CYP86A1 gene in cotton is specifically expressed in plant root. Wherein the CYP86A1-1 gene expression level is obviously related to the disease resistance. Compared with the susceptible upland cotton army cotton No.1, the gene has obviously enhanced expression and induced expression level in the disease-resistant H7124 root tissues of the island cotton. Based on PCR technology, obtaining a genome sequence and a full-length ORF sequence of CYP86A1-1 in Gossypium barbadense H7124, wherein the nucleotide sequence of the genome and the full-length cDNA ORF of the gene in the genome A subgroup (GbCYP86A1-1A) of the allopetraploid Gossypium barbadense H7124 is shown as SEQ ID NO. 1; the nucleotide sequence of the genome and the full-length cDNA ORF of the gene in the genome D subgroup (GbCYP86A1-1D) of the sea island cotton H7124 is shown as SEQ ID NO. 2. The gene does not contain introns, and the genome sequence of the coding region of the gene is identical with the full-length ORF sequence. Through the identification of verticillium wilt resistance, the gene is silenced in sea island cotton H7124 based on the VIGS technology to obviously reduce the verticillium wilt resistance of cotton, and the gene is overexpressed in arabidopsis thaliana to obviously improve the disease resistance.

Phylogenetic analysis, structural analysis and expression pattern analysis of CYP86 gene family in cotton

All CYP86 family genes were searched for in the complete genome of diploid Ramond cotton, all P450 superfamily genes in cotton were extracted according to the software HMMER3.0 and the Cytochrome P450(PF00067) domain protein family in the Pfam website, and these predicted family genes were validated with the SMART and INTERPROSCAN websites. Clustering analysis was performed on 11 CYP86 family genes in arabidopsis, and there were 10 CYP86 family genes in total in cotton.

The designation of the CYP86 gene family in arabidopsis thaliana was used as a reference, and the CYP86 family genes in cotton were systematically designated based on their homology, with 7 genes in the CYP86A class, 2 genes in the CYP86B class, and 1 gene in the CYP86C class in remmond cotton (fig. 1A). Wherein, the GrCYP86A1 has three homologous genes which are respectively named as GrCYP86A1-1, GrCYP86A1-2 and GrCYP86A 1-3; GrCYP86A7 has two homologous genes which are named GrCYP86A7-1 and GrCYP86A7-2 respectively; GrCYP86A8 has two homologous genes which are named GrCYP86A8-1 and GrCYP86A8-2 respectively; GrCYP86B1 also has two genes clustered to one branch with CYP86B1 in Arabidopsis, but the homology is not high, and the genes are tentatively named GrCYP86B1-1 and GrCYP86B1-2 (FIG. 1B). The structure of the family of genes was analyzed by website (GSDS)2.0(http:// GSDS. cbi. pku. edu. cn /) and the corresponding genomic sequence and its coding CDS sequence, of which only 3 genes have introns (FIG. 1C); the domain predictions using the SMART website all had a P450 core domain and a transmembrane domain, with the exception of GrCYP86A7-1 and GrCYP86A7-2 (FIG. 1D). In summary, we systematically classified and named the CYP86 family genes in cotton based on cluster analysis and homology analysis, analyzed the gene structure and Domain, and found that the CYP86 family genes are relatively conserved in structure.

To investigate the effect of CYP86 family genes in tetraploid cotton, the expression patterns of genes in vegetative organs (roots, stems, leaves), floral organs (petals, stamens, pistils), ovules (-3, 0, 3 days) and fibrous tissues (5, 10, 20, 25 days) were analyzed according to the transcriptome data of Gossypium hirsutum G.TM-1, and the FPKM values were log 2. As a result, the three homologous genes of the GhYP 86A1 class are specifically expressed in roots; while the ghcrypt 86A8 class is mainly expressed in floral organs, ovules and fibers; ghcrypt 86a7 is mainly expressed in stem, leaf, petal and 10-day fiber; GhYP 86B1-1 showed a constitutive expression pattern with high expression in various tissues, whereas GhYP 86B1-2 detected only subfamily A in tetraploids and was expressed only in floral organs and 25-day fibers; GhCYP86C1 was not substantially expressed in any tissue. The CYP86 family genes in cotton have conserved gene structure but diverse functions, and spatially distinct expression patterns may be associated with their function in plants (fig. 2A).

The functions of the related genes of the CYP86 family reported by the species are basically focused on the biosynthesis of lipid polyester, and mainly play roles in preventing water deficiency, preventing the influence of external biotic and abiotic stresses and enhancing the resistance of plants. In order to prove that CYP86 family genes act in a disease-resistant process, verticillium wilt pathogen strong virulence strain V991 is used for inducing the roots of cotton, three homologous genes with high expression in the roots and CYP86A1 and CYP86B1-1 are selected according to transcriptome, the change of expression level is detected at different time, and disease-resistant material sea island cotton H7124 and disease-susceptible material upland cotton military cotton No.1 are respectively used as resistance and sensitivity control. Among them, four genes all show higher expression in the disease-resistant material gossypium barbadense H7124, while three homologous genes of CYP86A1 class show a trend of significantly up-regulating expression in different materials and different periods of induction, especially the expression level is increased by several times in the late induction period of 144 hours in gossypium barbadense (FIG. 2B). Three homologous genes of GbCYP86A in sea island cotton are likely to sense the invasion of verticillium wilt bacteria to activate the corresponding disease-resistant immune process, so that the further colonization of pathogenic bacteria is prevented. The primers used in the study are listed in Table 1.

Table 1: specific primer for induction expression

Primer name	Primer sequence (5'-3')	Use of
			H2316F	CAGGGTGGAGCAGAAGATGTCTCT	CYP86A1-1 quantitative PCR
H2316R	GAGGTGGCAAATACAAAGTTAAGA	CYP86A1-1 quantitative PCR
			H2317F	GGAGCAAAAGATGTCTCTCACGC	CYP86A1-2 quantitative PCR
H2317R	TCCTCCATGCCTTTCCTTGTATCC	CYP86A1-2 quantitative PCR
			H2318F	CATGAAGCAAGGCCTTCGTGTTTA	CYP86A1-3 quantitative PCR
H2318R	CCACCAAAACCCCACTTGAACAA	CYP86A1-3 quantitative PCR
			H2320F	TCATTACAAACCGAAGACAAA	CYP86B1-1 quantitative PCR
H2320R	AATATCACGCAGGAACTTG	CYP86B1-1 quantitative PCR
			Y8991F	CGGTGGTGTGAAGAAGCCTCAT	Quantitative internal standard for cotton
Y8991R	AATTTCACGAACAAGCCTCTGGAA	Quantitative internal standard for cotton

(II) VIGS technology preliminarily identifies GbCYP86A1 type gene participating in disease resistance of cotton

As described above, the GbCYP86A1 gene may play an important role in the verticillium wilt resistance of cotton. In order to further research the effect of GbCYP86A1 genes in the disease-resistant process, one of three genes which silence GbCYP861A specifically in sea island cotton is respectively carried out by the VIGS technology, and 3 genes which silence simultaneously are selected from homologous fragments for preliminary functional identification. Specific segments of A3' UTR region are respectively selected, and four vectors of TRV2 GbCYP86A1-1, TRV2 GbCYP86A1-2, TRV2 GbCYP86A1-3 and TRV 2H 3091 are constructed to silence endogenous genes in H7124, wherein TRV1-TRV2 are used as control simulation treatment. To verify the feasibility of the VIGS system in cotton, both leaves and stems of plants were rendered albinism phenotype after silencing using the CLA1 gene encoding 1-deoxy-D-xylulose 5-phosphate synthase as reference (Gao et al, 2013). All constructed vectors were injected into cotyledons of cotton seedlings, about seven days in size, by the Agrobacterium-mediated method, and the uninjected seedlings were also grown in the same environment as a control. After two weeks all cotton leaves injected with TRV2-CLA1 vector exhibited a highly uniform albino phenotype (FIG. 3A). To further determine the silencing efficiency of the gene, RNA was extracted from each silencing strain and from the control strain, and gene expression was verified by real-time fluorescent quantitative pcr (qpcr). Silencing strains GbCYP86a1-1, GbCYP86a1-2, GbCYP86a1-3 and H3091 were expressed less than the uninjected control H7124 and the empty TRV2 vector, showing that all of these genes were significantly silenced at the transcriptional level, whereas the expression of two other homologous genes was not significantly affected by the specialized silencing of one of the genes (P <0.01) (fig. 3C).

After determining the silencing of the target gene, the disease resistance was identified in the two-leaf one-heart stage. Using cultured Verticillium dahliae V991, 25mL of 1X 10⁷The suspended spore liquid is used for grafting diseases in a mode of tearing the bottom and damaging the root. 24 plants were planted per material, with 3 replicates set. Watering is not carried out within three days after receiving diseases, and 50ml of water is supplemented every two days. The temperature and humidity in the room are ensured. The resistant variety H7124 and the susceptible variety Jun Cotton No.1 are respectively used as controls, cotyledons of Jun Cotton No.1 show obvious withering after 10 days of inoculation, and H7124 shows a little leaf yellowing phenotype after 15 days. Phenotypes after inoculation with verticillium wilt virus V99120 days and 25 days are shown (FIG. 3B). About 3 weeks after disease inoculation, the cotton seedlings of the silenced plants, especially TRV2: GbCYP86A1-1, TRV2: GbCYP86A1-2, and TRV2: H3091 leaves showed more wilting and yellowing, and the incidence of the three silenced materials was observed to be higher for 25 days and 30 days, while the disease-sensitive phenotype of TRV2: GbCYP86A1-1 and TRV2: H3091 was more severe (as shown in FIG. 4B); the same treated military cotton No.1 has the leaf blade basically fallen off at 25 days. In a word, the verticillium wilt sensitivity of H7124 can be remarkably improved by silencing GbCYP86A1-1 and 3 genes simultaneously, the sensitivity can be improved to a certain extent by silencing GbCYP86A1-2, but the silencing GbCYP86A1-3 shows that the functions of different homologous genes are differentiated, and the disease resistance response is different. Furthermore, we selected 24 plants per treatment for disease grade investigation, and found that only a small number of diseased leaves were investigated in the control TRV2:00 plants and the uninjected H7124, with a leaf disease rate of about 55% at 35 days. However, the diseased leaf rate of the silent gene GbCYP86A1-1 and the H3091 plant which silences 3 genes reaches 86 percent, and the silent gene GbCYP86A1-2 and GbCYP86A1-3 genesThe disease leaf rate of the plants is 77% and 69%, respectively, and the disease leaf rate of the disease-sensitive material army cotton reaches 100% (as shown in figure 4C).

Verticillium wilt is a soil-borne fungus, invades through the roots of plants and spreads throughout the body through vascular tissues. Verticillium wilt, when infected, blocks plant vascular tissue, giving it a brownish black color (Fradin and Thomma et al, 2010). The higher the infection degree, the darker the color, and the content of verticillium in vascular tissues can be visually shown by a stem splitting experiment. After 15 days of inoculation, the same parts (1 cm above cotyledonary node) of the silenced and control plants were subjected to oblique-cutting rod splitting experiments and observed by taking pictures under a body mirror. Also we observed that vascular tissue from plants silenced GbCYP86A1-1 and three genes simultaneously invaded more verticillium wilt, followed by plants silenced GbCYP86A1-2 and GbCYP86A1-3, whereas control plants and H7124 only invaded a small amount of hyphae, and water treated controls did not have any hyphae (FIG. 4D).

By comparing the disease conditions of plants with silent genes with those of a control group, the GbCYP86A1 gene endows the plants with resistance to verticillium wilt bacteria to different degrees (P < 0.01). Wherein GbCYP86A1-1 is an important gene of cotton for resisting verticillium wilt bacteria, has more obvious phenotype compared with other homologous genes, and has potential application value in cotton breeding for disease resistance. The primers used in the study are listed in Table 2.

Table 2: primers for amplification

Chromosomal location, evolutionary analysis, and localization of (tri) GbCYP86A 1-like genes

The GbCYP86 family genes are distributed on substantially different chromosomes in cotton tetraploids, with 3 homologous genes of the GbCYP86a1 family distributed on three chromosomes. GbCYP86A1-1 is on chromosome 3, GbCYP86A1-2 is on chromosome 7, and GbCYP86A1-3 is on chromosome 5. Meanwhile, more amino acid sequences of CYP86A1 genes in other species are obtained from NCBI databases and are respectively derived from plants such as sea island cotton, upland cotton, Ramond cotton, Arabidopsis, cocoa, poplar, grape, tobacco, sweet wormwood, sunflower, soybean, red bean, rice, corn and the like. It can be seen that GbCYP86a1 is widely present in different species. And drawing a phylogenetic tree through comparison between amino acid sequences, analyzing by adopting a MEGA5 software program, setting all parameters as defaults, and simultaneously applying a software DNAMAN for homology comparison. The result shows that CYP86A1 in each cotton variety has high homology and is relatively similar to the homology relation between cocoa and poplar. GFP4 subcellular localization vector was constructed, Agrobacterium was used to inject tobacco leaves, laser confocal microscopy was used to observe the subcellular localization of GbCYP86A1 genes, 3 genes were localized in the endoplasmic reticulum and membrane system connected to it as well as most of the P450 family genes (FIG. 5). Subcellular localization vectors are listed in table 3.

Table 3: recombinant primer for constructing subcellular localization vector

(IV) identification of disease resistance and phenotypic analysis after overexpression of GbCYP86A1 genes in arabidopsis thaliana

In order to further and deeply research the functions of GbCYP86A1 genes, three genes including GbCYP86A1-1, GbCYP86A1-2 and GbCYP86A1-3 are constructed on a 35 s-initiated overexpression vector PBI121, the three genes are transformed into an Arabidopsis wild type (Col-0) by an agrobacterium-mediated method, positive clones are screened on a resistance selection medium, DNA of positive plants is extracted for identification, and after the positive plants are screened for T3 generations, each gene finally identifies 6 homozygous transgenic line clones which are respectively named as OE1-OE 6. For 6 clones of each transgenic material, root RNA was extracted, overexpression of foreign genes was detected, expression characteristics were integrated, and two clones with highest expression, OE1 and OE2, were selected for further analysis (FIG. 6A).

First, the phenotypes of two clones of each transgenic line were observed, and when four true leaves of Arabidopsis were transferred from the medium to soil pots, no significant change in phenotype was observed after four weeks of cultivation (FIG. 7). Then inoculating verticillium dahliae V991 by root dipping method, the concentration of the suspension spore liquid is 1 multiplied by 10⁷spores/ml, disease development after two weeks, statistics of disease progression, calculation of disease index, and two weeks later root water-dip wild-type WT growth was normal as a control (fig. 6B). As a result, the disease index of wild type Arabidopsis WT reaches 72.5% after two weeks of inoculation, while the two clone disease indexes of Arabidopsis with overexpression of GbCYP86A1-1 gene are only 30% and 14%, and the verticillium wilt resistance is obviously enhanced; simultaneously, the two cloning diseases of Arabidopsis with over-expression of GbCYP86A1-2 gene are 42% and 55%, and the resistance is also improved; two cloning diseases of Arabidopsis thaliana overexpressing the GbCYP86A1-3 gene indicated that 66% was achieved, and no significant change in resistance was detected compared to the wild type (FIG. 6C). In order to detect the invasion condition of verticillium wilt bacteria, different clones of each transgenic line and the same parts of roots, stems and leaves of a control line WT are respectively taken 10 days after inoculation, the relative content of the verticillium wilt bacteria in tissues is respectively detected, each tissue is ground into powder and genome DNA is extracted, the content of fungi is measured by using a specific primer ITS-F based on a DNA transcription spacer region in ribosome and a specific reverse primer ST-Ve1-R of the verticillium wilt bacteria, and a cotton internal standard gene is used as a reference. The results showed that 10 days after inoculation, only a small amount of verticillium wilt bacteria was detected in the rhizome leaves, especially in the leaves, by both clones of transgenic Arabidopsis overexpressing the GbCYP86A1-1 gene. The relative content of verticillium wilt bacteria in tissues of plants overexpressed by GbCYP86A1-2 is also low, and the relative content of GbCYP86A1-3 in tissues of wild type WT is detected to be high (FIG. 6D). Analysis of the results shows that the early stage of infection with GbCYP86A1-1 and GbCYP86A1-2 overexpression lines show resistance, and only a small part of pathogenic fungi invade roots and colonize vascular bundles. We further designed experiments on the recovery of verticillium wilt bacteria invading stems at the early stage of infection and analyzedInfection rate of Arabidopsis thaliana. After 7 days of inoculation, 5 transgenic lines are randomly selected for each transgenic line, conventional tissue separation is carried out, 2cm stem segments are taken from the root nodes of the stems upwards, the surfaces of the stem segments are disinfected by 70% alcohol, sterile water is washed for 3-4 times, the stem segments are placed on a PDA culture medium at intervals, after the stem segments are cultured for 4 days at a constant temperature of 25 ℃, and after colonies of verticillium wilt bacteria grow out, the number of the colonies is observed and counted. The water-treated WT served as a control and no colonies grew. Two clones of transgenic plants overexpressing GbCYP86A1-3 and the stem node after wild-type WT inoculation clearly recovered more colonies, while transgenic plants overexpressing GbCYP86A1-1 recovered only particularly few colonies (FIG. 6E).

By combining the results, enough evidence proves that compared with GbCYP86A1-2 and GbCYP86A1-3, the GbCYP86A1-1 gene can obviously enhance the resistance of Arabidopsis to verticillium dahliae, and the disease resistance is mainly shown in the resistance to the initial stage of verticillium dahliae invasion, and can resist more verticillium dahliae invasion in the initial stage of infection, so that a small part of pathogenic bacteria colonize vascular tissues, and a more idea is provided for the research direction of plant disease resistance. The primers used in the study are listed in Table 4.

Table 4: enzyme digestion primer constructed by overexpression vector and quantitative detection primer

(V) observation of root structure of GbCYP86A1 gene after overexpression in Arabidopsis thaliana and transcriptome analysis of GbCYP86A1-1 transgenic Arabidopsis thaliana root

From two clones of each transgenic line, one clone with more obvious phenotype was selected for subsequent study, wherein the GbCYP86A1-1 transgenic line was selected to be OE2, and the GbCYP86A1-2 and GbCYP86A1-3 were selected to be OE1, respectively, and the over-expression number OEs is omitted in the later figure.

In order to further study the function of GbCYP86A1-1, different transgenic lines are vertically cultured, and the root length of seedlings growing for 8 days in a culture medium is investigated; simultaneously staining the cells with 50 mu g/mL Propidium Iodide (PI) for 30s, and observing and measuring the cell length in a fluorescence microscope; the condition of the root hairs was observed and counted using a stereoscope, 5 replicates per material, each replicate investigating at least 10 root hair lengths. The survey data is integrated, and statistical analysis shows that the root length, the root cell length and the root hair length of different transgenic materials and controls have no significant difference. It was shown that 3 transgenic Arabidopsis lines did not affect the growth of roots at the seedling stage of Arabidopsis (FIG. 8).

As Arabidopsis thaliana transformed with GbCYP86A1-1 gene shows particularly high resistance to verticillium wilt bacteria, in order to better analyze the functions of Arabidopsis thaliana at the whole genome level, wild type WT and GbCYP86A1-1 transgenic Arabidopsis thaliana which are 4 weeks old are respectively treated with water and verticillium wilt bacteria V991 for three days, root RNA is extracted, and transcriptome differential expression analysis is carried out.

Firstly, two materials treated by water are compared and analyzed, expression difference genes are screened according to a sequencing result, and the genes with the difference multiple of more than 1.5 times are selected for GO enrichment. The results show that genes related to stress, biostimulation, defense reaction, plant immunity, redox, secondary metabolism, cell wall structure and the like are significantly enriched in the roots of the transgenic arabidopsis thaliana GbCYP86a1-1 (fig. 9A).

Aiming at structural resistance related items enriched by transcriptome, including secondary metabolism, lipid transport, flavonoid synthesis in the phenylpropane metabolism process and the external structure of a cell wall, the gene GbCYP86A1-1 may participate in changing the lipid metabolism and the cell wall structure of Arabidopsis thaliana so as to achieve the purpose of resisting verticillium dahliae. Therefore, we performed microscopic observation of a cross section of 4-week-old roots of Arabidopsis thaliana at maturity, sampled 10mm from 20mm from the base of the main root (FIG. 9B), made a semi-thin section, fixed to a microtome and cut into 2.0 μm thick sections at the middle position by fixing, dehydrating and embedding with resin, stained the cells with lipophilic dye Sudan 7B (0.1%, w/v; 50% (v/v) PEG400, 45% (v/v) glycerol, 5% water), washed with SDS (1%) at room temperature for 1 hour, and then washed with water and placed in glycerol: microscopic observation in water (1: 1). As a result, it was found that more ester-like substances were accumulated on the cell wall of the outer cell of the root of GbCYP86A1-1 transgenic Arabidopsis thaliana, the red color was more distinct, and the cell structure was more compact and dense, which may be a key cause for improving disease resistance, compared with wild type WT (FIG. 9C). We further detect the relative content of fatty acid in different transgenic plant roots, firstly preparing external standards with different concentrations, drawing a standard curve, then fully grinding the sample, carrying out corresponding depolymerization reaction and methyl esterification treatment, taking 10 mu g of dotriacontane as an internal standard, and applying a gas chromatograph to carry out the relative content determination. As a result, the relative content of C16-C18 fatty acids in roots of GbCYP86A1-1 transgenic Arabidopsis was significantly higher than that of other lines (FIG. 9D). Comprehensive result analysis GbCYP86A1-1 gene influences fatty acid metabolism of root cells of arabidopsis thaliana and lipid content of suberect spatulate cells, so that a structural barrier is formed at the root to resist the invasion of most pathogenic fungi.

The biological function of CYP86a1 has been studied in arabidopsis thaliana, as a type of hydroxylase, located in the endoplasmic reticulum and mainly involved in the hydroxylation of fatty acids, which undergo a series of redox reactions and elongation of fatty acid chains to finally polymerize into complex biopolyesters, which need to be exported from the endoplasmic reticulum and pass through the cytoplasmic membrane, and then aggregate to the cell wall to form suberin. Studies have now shown that Lipid Transfer Proteins (LTPs) and ATP-binding cassette (ABC) transporters are involved in transmembrane transport of suberin components (vishwatath et al, 2015). Through transcriptome analysis, we enriched some LTP transporters to participate in transmembrane transport, and also enriched a large number of ABC transporters and AAA-ATPases, which are genes with ATPase activity, and play a role in energy supply. These genes all show remarkably high expression in transgenic plants, and because the overexpression of the GbCYP86A1-1 gene promotes the lipid polymerization and transportation in plants, the LTP transporter is required to support transmembrane transport and the ABC transporter provides required energy. Meanwhile, a plurality of genes participating in the secondary metabolic process are also enriched, the genes participate in the phenylpropane metabolic process or the cork synthesis process, and the expression in a transgenic line is also obviously improved. For example, Phenylalanine Ammonia Lyase (PAL) is an enzyme that catalyzes the step 1 reaction process of the phenylalanine metabolic pathway and is also the rate-limiting enzyme in this process, and both 1 aminocyclopropane-1-carboxylic Acid (ACC) oxidase and PAL play important roles in the healing process of lesions (Kato et al, 2000). The KCS gene encodes β -Ketoester CoA Synthetase (KCS), a key enzyme in fatty acid elongation processes (joubs et al, 2000). The P450 is shown to be widely involved in the phenylpropane metabolic process and the synthesis process of secondary metabolites, and provides a reference candidate gene for the research of the biosynthesis process of the suberin.

Possible regulators of suberin synthesis are located in the WRKY-, NAC-, MYB-specific transcription factor regions, which are preferentially expressed in suberized tissues (Kilian et al, 2000). The course-associated (ethylene-responsive) (AP2/ERF) transcription factor is involved in secondary cell wall modification (Lasserre et al, 2000). However, direct evidence that these transcription factors are involved in the regulation of the suberization process is not sufficient and further intensive studies are required. In the transcriptome, we also found many WRKY transcription factors and ERF transcription factors, and also found many receptor-like protein kinases (RLKs), receptor-like proteins (RLPs) and disease course-related Proteins (PRs), and all showed high expression in GbCYP86A1-1 transgenic line, presumably because some signal molecules are influenced by lipid content change and cell wall modification, and further activated root induced immune process (FIG. 10). RLKs and RLPs are positioned in cell walls and plasma membranes and are possibly highly expressed under the influence of structural changes of root cell walls, signals possibly influence WRKY transcription factors and ERF transcription factors, the WRKY transcription factors have been researched for a plurality of functions in pathogenic bacteria stress, and the disease course related ERF transcription factors can be specifically combined with a disease course related protein promoter element to regulate and control the expression of disease course related protein genes, so that the increase of the expression of PR genes regulated by the transcription factors can be another important reason for improving the disease resistance of plants. The enhancement of the plant structure resistance can also influence the change of receptors in cells, so that a responsive disease-resistant immune pathway is activated, and the expression of a disease-resistant gene PR gene is finally influenced by the regulation and control of a transcription factor, so that the plant structure resistance has an important significance in the research of resisting pathogenic fungi, and a new idea is provided for deeply excavating and researching disease-resistant candidate genes.

We compared the differential gene after three days of GbCYP86A1-1 transgenic Arabidopsis treatment and water treatment and the differential gene expression between the treatment of the disease in WT and the water treatment, respectively, and performed GO enrichment analysis. Firstly, obvious difference is shown from the number of difference genes, 2541 difference genes are totally arranged between disease treatment and water treatment in roots of GbCYP86A1-1 transgenic arabidopsis thaliana, only 277 genes with more than 1.5-fold difference exist, and only a few genes are obviously changed; however, there were 3616 different genes in wild-type WT, and among them, the number of genes more than 1.5-fold different was as much as 1515, which was more than 5-fold higher than that in the transgenic line. Due to improvement of disease resistance, most verticillium wilt germs cannot invade or colonize, the root of a transgenic line is changed slightly after being infected, the injury is small, internal genes basically tend to be stable, and GO is enriched into some items related to growth and development; the relative variation of wild type was large, mainly due to the severe effects of verticillium wilt pathogen invading roots on root cell tissue, while the resistant genes needed to be expressed to maintain normal growth and development, and therefore enriched in many entries, including growth and development related and resistance related entries (FIG. 11A). Meanwhile, we observed the root tissue in a free-hand section, and after selecting the roots of 4-week-sized plants and treating the roots with verticillium wilt for three days, we observed the fixed longitudinal section, and compared with the other two transgenic materials and wild type WT, the root of GbCYP86A1-1 transgenic Arabidopsis line has almost no invasion of black mycelium, which is also identical with the result of transcriptome (FIG. 11B). Pathogenic bacteria in the root are difficult to invade and colonize, so that the influence on the whole root gene is small; in contrast, wild type plants are damaged internally due to severe root infection, so that more genes are affected in cells, and some resistance genes need to be activated to prevent further propagation of pathogenic bacteria.

To verify the accuracy of transcripts, transcription was performedPerforming related qRT-PCR verification on the group sequencing result, selecting 30 genes with the multiple of difference more than 1.5 times of the transcriptome sequencing result after water treatment for verification, performing statistical analysis on the results of the water treatment and the disease treatment, drawing a standard curve, and calculating R²Value check fit (FIG. 12).

The gene related to the secondary metabolic process and the disease-resistant immune process in GbCYP86A1-1 transgenic Arabidopsis and wild Arabidopsis WT was verified by quantitative PCR, and the results matched the transcriptome results (FIG. 13).

The comprehensive result shows that the GbCYP86A1-1 gene can obviously increase the resistance of arabidopsis thaliana to verticillium wilt, the resistance is mainly embodied in that the invasion of root verticillium wilt is influenced, and the disease resistance is endowed to plants by influencing the physical structure of root cell walls of arabidopsis thaliana and inducing the activation of a disease-resistant channel.

(six) GbCYP86A1-1 can induce the expression of disease course related protein of cotton roots

From the transcriptome results we found that there were many increased expression of the downstream PR genes in the transgenic lines, as previously reported that PR genes had a very important role in both signal recognition and plant immunity (Mcfadd et al, 2001; Sels et al, 2008). In order to identify a plurality of PR genes related to cotton defense response mechanisms, the expression patterns of PR1, PR2, PR3, PR4, PR5, PR6, PR9 and PR16 in H7124 cotton and cotton military under the induction of verticillium wilt are analyzed by combining the results of transcriptome, and the PR genes are remarkably subjected to induced expression change in two cotton species and have basically consistent induction trend. To investigate whether silencing of the GbCYP86a1-1 gene affected defense-related genes in roots, we further analyzed the expression of PR genes in silenced plants. When the GbCYP86a1-1 gene is specifically silenced, the expression levels of PR1, PR5 and PR16 are all significantly reduced, and PR2 and PR4 are significantly reduced, which may also be one of the causes of the reduction of plant disease resistance (fig. 14). The amplification primers used for the quantitative analysis are listed in Table 5.

Table 5: primers for amplification

Primer name	Primer sequence (5'-3')	Use of
			GbPR1F	AAGAATGTGGGTTAGTGAGAGGGT	GbPR1 quantitative PCR
GbPR1R	ACCACTTGAGTATAATGCCCGC	GbPR1 quantitative PCR
			GbPR2F	CCACCAGCAGCAGAAGTTATCG	GbPR2 quantitative PCR
GbPR2R	TTCAAGGTTTGCACTCGGAAGA	GbPR2 quantitative PCR
			GbPR3F	ACTCCACAATCACCGAAGCCAT	GbPR3 quantitative PCR
GbPR3R	GCATTCCAACCCTTACCACATTC	GbPR3 quantitative PCR
			GbPR4F	TTGCGGCAATGGCTTCAATC	GbPR4 quantitative PCR
GbPR4R	TGCTCTCACATTATTCGGCA	GbPR4 quantitative PCR
			GbPR5F	GCCGTGATTCATACAGTTATCCTCA	GbPR5 quantitative PCR
GbPR5R	TTGGCTCTTACTTCCGACCATCT	GbPR5 quantitative PCR
			GbPR6F	CTGGGTGTCCTGGGAAGAAC	GbPR6 quantitative PCR
GbPR6R	TTGTAGGGGGACGAACAACG	GbPR6 quantitative PCR
			GbPR9F	CAACAGCGCCAACATACAGAG	GbPR9 quantitative PCR
GbPR9R	CAGCACAAGAGACAATGCCAG	GbPR9 quantitative PCR
			GbPR16F	CCCAAAGCTTGCCAAAGCA	GbPR10 quantitative PCR
GbPR16R	GAACATTGGCTGGAGTGACC	GbPR10 quantitative PCR

(seventh) GbCYP86A1-1 influences the resistance of the cell wall structure of the plant and induces the activation of the disease-resistant signal path

The GbCYP86A1-1 gene can be up-regulated and expressed after being induced by verticillium dahliae, the main function of the gene is to promote the hydroxylation of fatty acid in endoplasmic reticulum so as to activate a series of lipid metabolic processes in vivo, the physical structure resistance of plant root cell walls is increased through the polymerization of lipid and the transmembrane transport of lipid transporter LTP, and ATPase is required to provide a large amount of energy in the process. While inducing structural changes such as cell wall thrombosis and cell wall modification, some signal molecules are also excited, and the overall immune mechanism inside plant roots is enhanced and activated by gradually transmitting and finally regulating the expression of PR genes (figure 15). The research is expected to improve the expression of GbCYP86A1-1 gene by using GbCYP86A1-1 as a target gene through gene engineering technologies such as transgenosis and the like, and culture a new germplasm with remarkably improved resistance and apply the new germplasm in production.

Sequence listing

<110> Nanjing university of agriculture

<120> cotton verticillium wilt resistant gene GbCYP86A1-1 and application thereof

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cttcgagcaa caggtggttc agccacctat caaacatgta ccattgctat tccatttttg 240

gctcgcaagc aagggttcta cactgtcact tgtcacccca aaaacattga gcacgttctc 300

cggacccggt tcgataatta ccccaaaggg cctcattggc aggctgcatt tcatgatctt 360

ttggggcaag ggatcttcaa tagtgatgga gaatcgtggc tgattcagag gaaaacagca 420

gcacttgagt tcacgaccag gacactcagg caagccatgg gtcgttgggt taataggact 480

atcaagaatc gtctatggtg tattttggac aaagcatcaa atgagaagaa agcagtggat 540

ttgcaagact tgttgcttcg tttaaccttt gacaacattt gtgggcttac atttggcaaa 600

gacccaaaaa cactttctca cgagttacca gataaccctt ttgctactgc tttcgataca 660

gccactgaag caactcttaa taggcttctt taccctggtt tactatggag attgaagaag 720

attttgggga taggagctga gaaaagatta aagagtagtc tccgaatcgt tgaaaactat 780

atgaacgagg ccattgaagc acgaaaagaa gctccttcgg atgatctact gtcccgtttc 840

atgaaaaaaa aagatgctgg gggaaacctt ttcacaagca ccgttcttca acgcatcgct 900

ctgaacttcg tcctcgctgg ccgtgacacc tcttccgtag ccctcagctg gttcttctgg 960

ctcgtaatga accacccaga gattgagcaa aagatcatca atgaaatatc aagggttctc 1020

cgcaacaccc gcggcccaga tactaagaaa tggatggaag agccactgat gttcgatgaa 1080

gcagacaagc tgatatatct gaaagcagca ttagctgaaa cactgcggtt atacccctcg 1140

gttcctcagg acttcaagta cgtggtcgaa gacgatgtgt taccagatgg cacattggtt 1200

cccgctggct ccaccgtcac atattcgata tactcagttg gaagaatgaa gagtatatgg 1260

ggagaagatt gcatggagtt taagcccgaa agatggctat cagcagaagg tgacaagttc 1320

gaggcaccaa aggatggtta caagtttgtg gcgttcaacg ctggaccaag gacttgtttg 1380

ggcaaagact tggcttactt gcaaatgaag tcggtggcct ccgcagttct gctgcgttat 1440

cgggtttcgc tggttcctgg acacagggtg gagcagaaga tgtctctcac actgtttatg 1500

aagaaaggtc ttcgtgttta cttgcagccg cgtctacttg catag 1545

<210> 2

<211> 1545

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggaattgg aaaaccctcc atttgggttc accctagcag ctgctgcaac atttgcttat 60

gcaatatggt tctatctgct agccaagagg ttaagtggtc caagggtatg gcctctggtc 120

ggtagcctac catttctttt catgaaccgg aggaggatgc atgattggat tgctagtaat 180

cttcgagcaa cgggtggttc agccacctat caaacatgta ccattgctgt tccatttttg 240

gctcgcaagc aagggttcta cactgtcact tgtcacccca aaaacattga gcacattctc 300

cggacccggt tcgataatta ccccaaaggg cctcattggc aggctgcatt tcatgatctt 360

ttggggcaag ggatcttcaa cagtgatgga gaatcgtggc tgatacagag gaaaacagca 420

gcacttgagt tcacgaccag gacactcagg caagccatgg gtcgttgggt taataggact 480

atcaagaatc gtctatggtg tattttggac aaagcatcaa atgagaagaa agcagtggat 540

ttgcaagact tgttgcttcg tttaaccttt gacaacattt gtgggcttac atttggcaaa 600

gacccacaaa cactttctca cgagttacca gataaccctt ttgctactgc tttcgataca 660

gccactgaag caactcttaa taggcttctt taccctggtt tactatggag attgaagaag 720

attttgggga taggagctga gaaaagatta aagagtagcc tccgaatcgt tgaaaactac 780

atgaacgagg ccattgaagc acgaaaagaa gctccattgg atgatctact gtcccgtttc 840

atgaagaaaa aagatgctgg gggaaacctt ttcacaagca ccgttcttca atgcatcgct 900

ctgaacttcg tcctcgctgg tcgtgacacc tcttccgtag ccctcagctg gttcttctgg 960

ctcgtaatga accacccaga gattgagcaa aagatcatcg atgaaatatc aagggttctc 1020

cgcaacaccc gcggcccaga taccaagaaa tgggtggaag agccactgat gttcgatgaa 1080

gcagacaagc tgatatatct gaaagcagca ttagctgaaa cactgcggtt atacccctcg 1140

gttcctcagg acttcaagta cgttgtcgaa gacgatgtgt taccagatgg cacattggtt 1200

cccgctggct ccaccgtcac atattcaata tactcagttg gaagaatgaa gagtatatgg 1260

ggagaagatt gcatggagtt taagcccgaa agatggctat cagcagaagg tgacaagttc 1320

gaggcaccaa aggatggtta caagtttgtg gcgttcaacg ctggaccaag gacttgtttg 1380

ggcaaagact tggcctactt gcaaatgaag tcggtggcct ccgcagttct gctgcgttat 1440

cgggtttcgc tggttcctgg acacagggtg gagcagaaga tgtctctcac actgtttatg 1500

aagaaaggtc ttcgtgttta cttgcagccg cgtctacttg catag 1545

<210> 3

<211> 514

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Glu Leu Glu Asn Leu Pro Phe Gly Phe Thr Leu Ala Ala Ala Ala

1 5 10 15

Thr Phe Ala Tyr Ala Ile Trp Phe Tyr Leu Leu Ala Lys Arg Leu Ser

20 25 30

Gly Pro Arg Val Trp Pro Leu Val Gly Ser Leu Pro Phe Leu Phe Met

35 40 45

Asn Arg Arg Arg Met His Asp Trp Ile Ala Ser Asn Leu Arg Ala Thr

50 55 60

Gly Gly Ser Ala Thr Tyr Gln Thr Cys Thr Ile Ala Ile Pro Phe Leu

65 70 75 80

Ala Arg Lys Gln Gly Phe Tyr Thr Val Thr Cys His Pro Lys Asn Ile

85 90 95

Glu His Val Leu Arg Thr Arg Phe Asp Asn Tyr Pro Lys Gly Pro His

100 105 110

Trp Gln Ala Ala Phe His Asp Leu Leu Gly Gln Gly Ile Phe Asn Ser

115 120 125

Asp Gly Glu Ser Trp Leu Ile Gln Arg Lys Thr Ala Ala Leu Glu Phe

130 135 140

Thr Thr Arg Thr Leu Arg Gln Ala Met Gly Arg Trp Val Asn Arg Thr

145 150 155 160

Ile Lys Asn Arg Leu Trp Cys Ile Leu Asp Lys Ala Ser Asn Glu Lys

165 170 175

Lys Ala Val Asp Leu Gln Asp Leu Leu Leu Arg Leu Thr Phe Asp Asn

180 185 190

Ile Cys Gly Leu Thr Phe Gly Lys Asp Pro Lys Thr Leu Ser His Glu

195 200 205

Leu Pro Asp Asn Pro Phe Ala Thr Ala Phe Asp Thr Ala Thr Glu Ala

210 215 220

Thr Leu Asn Arg Leu Leu Tyr Pro Gly Leu Leu Trp Arg Leu Lys Lys

225 230 235 240

Ile Leu Gly Ile Gly Ala Glu Lys Arg Leu Lys Ser Ser Leu Arg Ile

245 250 255

Val Glu Asn Tyr Met Asn Glu Ala Ile Glu Ala Arg Lys Glu Ala Pro

260 265 270

Ser Asp Asp Leu Leu Ser Arg Phe Met Lys Lys Lys Asp Ala Gly Gly

275 280 285

Asn Leu Phe Thr Ser Thr Val Leu Gln Arg Ile Ala Leu Asn Phe Val

290 295 300

Leu Ala Gly Arg Asp Thr Ser Ser Val Ala Leu Ser Trp Phe Phe Trp

305 310 315 320

Leu Val Met Asn His Pro Glu Ile Glu Gln Lys Ile Ile Asn Glu Ile

325 330 335

Ser Arg Val Leu Arg Asn Thr Arg Gly Pro Asp Thr Lys Lys Trp Met

340 345 350

Glu Glu Pro Leu Met Phe Asp Glu Ala Asp Lys Leu Ile Tyr Leu Lys

355 360 365

Ala Ala Leu Ala Glu Thr Leu Arg Leu Tyr Pro Ser Val Pro Gln Asp

370 375 380

Phe Lys Tyr Val Val Glu Asp Asp Val Leu Pro Asp Gly Thr Leu Val

385 390 395 400

Pro Ala Gly Ser Thr Val Thr Tyr Ser Ile Tyr Ser Val Gly Arg Met

405 410 415

Lys Ser Ile Trp Gly Glu Asp Cys Met Glu Phe Lys Pro Glu Arg Trp

420 425 430

Leu Ser Ala Glu Gly Asp Lys Phe Glu Ala Pro Lys Asp Gly Tyr Lys

435 440 445

Phe Val Ala Phe Asn Ala Gly Pro Arg Thr Cys Leu Gly Lys Asp Leu

450 455 460

Ala Tyr Leu Gln Met Lys Ser Val Ala Ser Ala Val Leu Leu Arg Tyr

465 470 475 480

Arg Val Ser Leu Val Pro Gly His Arg Val Glu Gln Lys Met Ser Leu

485 490 495

Thr Leu Phe Met Lys Lys Gly Leu Arg Val Tyr Leu Gln Pro Arg Leu

500 505 510

Leu Ala

<210> 4

<211> 514

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Glu Leu Glu Asn Pro Pro Phe Gly Phe Thr Leu Ala Ala Ala Ala

1 5 10 15

Thr Phe Ala Tyr Ala Ile Trp Phe Tyr Leu Leu Ala Lys Arg Leu Ser

20 25 30

Gly Pro Arg Val Trp Pro Leu Val Gly Ser Leu Pro Phe Leu Phe Met

35 40 45

Asn Arg Arg Arg Met His Asp Trp Ile Ala Ser Asn Leu Arg Ala Thr

50 55 60

Gly Gly Ser Ala Thr Tyr Gln Thr Cys Thr Ile Ala Val Pro Phe Leu

65 70 75 80

Ala Arg Lys Gln Gly Phe Tyr Thr Val Thr Cys His Pro Lys Asn Ile

85 90 95

Glu His Ile Leu Arg Thr Arg Phe Asp Asn Tyr Pro Lys Gly Pro His

100 105 110

Trp Gln Ala Ala Phe His Asp Leu Leu Gly Gln Gly Ile Phe Asn Ser

115 120 125

Asp Gly Glu Ser Trp Leu Ile Gln Arg Lys Thr Ala Ala Leu Glu Phe

130 135 140

Thr Thr Arg Thr Leu Arg Gln Ala Met Gly Arg Trp Val Asn Arg Thr

145 150 155 160

Ile Lys Asn Arg Leu Trp Cys Ile Leu Asp Lys Ala Ser Asn Glu Lys

165 170 175

Lys Ala Val Asp Leu Gln Asp Leu Leu Leu Arg Leu Thr Phe Asp Asn

180 185 190

Ile Cys Gly Leu Thr Phe Gly Lys Asp Pro Gln Thr Leu Ser His Glu

195 200 205

Leu Pro Asp Asn Pro Phe Ala Thr Ala Phe Asp Thr Ala Thr Glu Ala

210 215 220

Thr Leu Asn Arg Leu Leu Tyr Pro Gly Leu Leu Trp Arg Leu Lys Lys

225 230 235 240

Ile Leu Gly Ile Gly Ala Glu Lys Arg Leu Lys Ser Ser Leu Arg Ile

245 250 255

Val Glu Asn Tyr Met Asn Glu Ala Ile Glu Ala Arg Lys Glu Ala Pro

260 265 270

Leu Asp Asp Leu Leu Ser Arg Phe Met Lys Lys Lys Asp Ala Gly Gly

275 280 285

Asn Leu Phe Thr Ser Thr Val Leu Gln Cys Ile Ala Leu Asn Phe Val

290 295 300

Leu Ala Gly Arg Asp Thr Ser Ser Val Ala Leu Ser Trp Phe Phe Trp

305 310 315 320

Leu Val Met Asn His Pro Glu Ile Glu Gln Lys Ile Ile Asp Glu Ile

325 330 335

Ser Arg Val Leu Arg Asn Thr Arg Gly Pro Asp Thr Lys Lys Trp Val

340 345 350

Glu Glu Pro Leu Met Phe Asp Glu Ala Asp Lys Leu Ile Tyr Leu Lys

355 360 365

Ala Ala Leu Ala Glu Thr Leu Arg Leu Tyr Pro Ser Val Pro Gln Asp

370 375 380

Phe Lys Tyr Val Val Glu Asp Asp Val Leu Pro Asp Gly Thr Leu Val

385 390 395 400

Pro Ala Gly Ser Thr Val Thr Tyr Ser Ile Tyr Ser Val Gly Arg Met

405 410 415

Lys Ser Ile Trp Gly Glu Asp Cys Met Glu Phe Lys Pro Glu Arg Trp

420 425 430

Leu Ser Ala Glu Gly Asp Lys Phe Glu Ala Pro Lys Asp Gly Tyr Lys

435 440 445

Phe Val Ala Phe Asn Ala Gly Pro Arg Thr Cys Leu Gly Lys Asp Leu

450 455 460

Ala Tyr Leu Gln Met Lys Ser Val Ala Ser Ala Val Leu Leu Arg Tyr

465 470 475 480

Arg Val Ser Leu Val Pro Gly His Arg Val Glu Gln Lys Met Ser Leu

485 490 495

Thr Leu Phe Met Lys Lys Gly Leu Arg Val Tyr Leu Gln Pro Arg Leu

500 505 510

Leu Ala

Claims (3)

1. As shown in SEQ ID NO.1 or SEQ ID number 2GbCYP86A1-1The application of the gene in improving the disease resistance of cotton and breeding new cotton germplasm is characterized in that: as described inGbCYP86A1-1The gene is used as target gene, and is overexpressed by genetic engineering methodGbCYP86A1-1Genes, the novel cotton germplasm with remarkably improved verticillium wilt resistance is cultivated and applied to production.

2. A method for improving verticillium wilt resistance of cotton is characterized by comprising the following steps: overexpression of nucleotide sequence shown as SEQ ID NO.1 or SEQ ID number 2 in cottonGbCYP86A1-1A gene.

3. Contains nucleotide sequence shown as SEQ ID NO.1 or SEQ ID number 2GbCYP86A1-1A recombinant vector of the gene,The expression cassette, transgenic cell line or recombinant strain are used in raising the disease resistance of cotton verticillium wilt and breeding new cotton variety.

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