CN109402147A - The gene GbCYP86A1-1 of anti-cotton verticillium wilt and its application - Google Patents

Abstract

The present invention discloses gene GbCYP86A1-1 and its application of a kind of anti-cotton verticillium wilt, belongs to biological technology application.GbCYP86A1-1 gene of the present invention encodes a fatty acid ω-hydroxylase.The present invention provides GbCYP86A1-1 in the genome A subgroup in allotetraploid sea island cotton H7124, genome and overall length ORF nucleotide sequence (the gene intronless) and amino acid sequence in D subgroup.CYP86A1-1 gene in the cotton specifically expressing in root is induced significant up-regulated expression by verticillium wilt pathogen.Compared with susceptible Upland Cotton army cotton No. 1, in disease-resistant island cotton variety H7124 root tissue, expression and induced expression level significantly increase the gene.By verticillium wilt pathogen Resistance Identification, based on VIGS technology, the silencing gene can significantly reduce the resistance to verticillium wilt of cotton in sea island cotton H7124, and by the gene, overexpression is remarkably improved the disease resistance of transgenic arabidopsis in arabidopsis.

Description

The gene GbCYP86A1-1 of anti-cotton verticillium wilt and its application

Technical field

The invention belongs to biological technology application, it is related to a kind of cotton GbCYP86A1-1 gene and its application, the gene Encode a fatty acid ω-hydroxylase.By to the CYP86 subfamily member in the cytochrome P450 gene family in cotton Network analysis is carried out, the target gene that can significantly improve cotton and arabidopsis resistance to verticillium wilt is excavated.In cotton CYP86A1-1 gene specifically expressing in root is induced significant up-regulated expression by verticillium wilt pathogen.With susceptible Upland Cotton army cotton 1 It number compares, in disease-resistant island cotton variety H7124 root tissue, expression and induced expression level significantly increase the gene.Benefit The amino acid sequence of the genome sequence of the gene, overall length ORF sequence and coding is obtained in sea island cotton H7124 with round pcr. It is studied using disease resistance and function of the biotechnology to the gene.By verticillium wilt pathogen Resistance Identification, it is based on VIGS technology The silencing gene can significantly reduce the resistance to verticillium wilt of cotton in sea island cotton H7124, which is crossed scale in arabidopsis Up to the disease resistance for being remarkably improved transgenic arabidopsis.

Background technique

Plant shows the special energy of various defence pathogens during with the long-term interaction of pathogen, coevolution Power, this defence capability are known as disease resistance on pathology.For a long time, people are to respectively observed phenomenon, from difference Angle disease resistance is described and is classified.The disease resistance that plant shows after pathogen is invaded mainly passes through two ways Diameter.One side is that the chemical component that certain institutional frameworks of plant itself are changed or synthesized has disease-resistant function, Qian Zhezhu There are bolt matter, cutin, wax and the other some special structures etc. of cell wall, referred to as structural resistance.On the other hand work as After pathogen breaches the intrinsic defensive barrier of plant, a series of disease resistance response that plant can show pathogen Physiology and biochemistries comes Cope with germ invasion, that is, Physiology and biochemistry resistance (Conrath et al., 2002).

The institutional framework resistance of plant can be divided into two classes: the structural resistance of intrinsic structural resistance and induction.The study found that Its institutional framework of the cotton variety of different resistances has differences, especially the difference between vascular bundle.Unit area in disease-resistant variety Cell quantity one times or more more than susceptible variety, disease-resistant variety xylem compared with susceptible variety is solid and intercellular gap compared with Cellule wall is thicker, and vessel cell arranges more closely, and the deposition of callose significantly increases, and the bacterium amount of pathogen is then obvious low In susceptible variety, which improves the mechanical disease-resistant performance of cotton, reduces the success rate of verticillium wilt pathogen invasion, so that Germ be difficult to extend in plant body (Zhang et al., 2013；Mace et al.,2010).The structural resistance of induction refers to Plant by, by complicated molecular regulation mechanism, inducing a series of intracorporal metabolic alterations of plant after the induction of pathogen, Occur the change of morphosis on cellular level, to resist the invasion and extension of external pathogen, finally resists plant Property.The resistance of induction is mainly reflected in bolt matter, lignifying, conduit parenchymal tissue hyperplasia and the colloid of cell wall and tissue Or formation of thylose etc. (Bell et al., 1967).Numerous studies are reported, after pathogen infection plant, will form a large amount of glue Substance, so that the invasion of pathogen is hindered, in disease plant, it can be observed that there are thylose or colloid, leaves in conduit Arteries and veins parenchyma cell is twisted into irregular shape, and duct wall thickens, root there are a large amount of polysaccharide materials (Daayf et al., 1997)。

The physiological metabolism of plant changes under the induction of pathogen, and various physiological acoustic signals occur in plant body, Such as the accumulation of phytoalexin, the outburst of active oxygen, the variation of plant defense enzyme system and the table of PR gene It reaches.In pathogen infection plant, inducer makes host plant build up a kind of low molecular weight antibiotic property secondary metabolite, Referred to as phytoalexin, the generation and accumulation of phytoalexin, be an important factor for so that plant is encroached on from some germs it One and plant disease-resistant chemical fundamentals.Plant generates active oxygen, belongs to the disease resistance response of earliest period.Host plant is inducing A large amount of active oxygen is generated under the action of object in short-term, levels of reactive oxygen species has direct antibacterial action, can kill disease Opportunistic pathogen.But, the active oxygen of high concentration can injure plant cell, and activity keto concentration need to be carried out by the intracorporal protective enzyme of plant It is appropriate to adjust.Meanwhile active oxygen also passes through the degree of lignification, hydroxyproline glycoprotein for enhancing host plant histocyte wall Activity etc. that is oxidation cross-linked and adjusting protective enzyme improves its ability (Alvarez et al., 2008) for resisting pathogen invasion. Cotton by verticillium wilt pathogen when being infected, peroxidase (ROD), superoxide dismutase (SOD), glutathione S-transferase (GSTs), phenylalanine lyase (PAL) and polygalacturonase inhibitory protein (PGIP) etc. be anti-oxidant, resistant to hydrolysis enzyme Timely regulating and expressing is understood, to slow down and inhibit verticillium wilt pathogen to degradation (the James and Dubery et of plant cell wall al.,2001).Pathogenesis-related proteins (PRs) by pathogen coerce and inducing expression, be plant generated under pathology environment one Class can directly attack the albumen of pathogen.The inducing expression of pathogenesis-related proteins is considered as the life of certain plants disease resistance response Change one of index.

There is complicated and perfect resistance signals to transmit network in plant.Although research is not also very thoroughly It is thorough, but scientist summarizes according to the result of study of plant innate immune system and proposes classical zipper mode, The seasaw battle of plant and pathogen has also embodied the importance of innate immune system, the door that can refuse a large amount of pathogen Outside (Jones and Dangl et al., 2006).Some cell membrane surface receptors play emphatically in the transmittance process of signal It acts on.Some researches show that ISO1 receptor albuminoid kinases can be interacted in a manner of ligand with FLS2 and ERF, regulation disease Former associated molecular pattern (PAMP) triggers immune (the Chen et al., 2014) of plant.RLP30, SOBIRl, BAKl facilitate Resist septic fungi, rlp30-2, sobirl1-12, bakl-5 mutant (Zhang et sensitive to soybean pyrenomycetes and botrytis cinerea al.,2013).Transcription factor family relevant to defense signaling mainly has WRKY family, bZIP family, ERF class in plant Transcription factor and MYB class transcription factor.WRKY transcription factor can positively or negatively regulate and control Plant defense responses, such as: water The induction of OsWRKY45 and OsWRKY23 gene response Pyricularia oryzae in rice, enhancing is to false unit cell after being overexpressed in arabidopsis Bacterium resistance (Qiu et al., 2009；Jing et al., 2009), play positive regulation in disease resistance of plant； Plant is more susceptible after AtWRKY38 and WRKY62 gene is overexpressed in arabidopsis, and the single mutant of wrky38 or wrky62 or The bis- deletion mutants of wrky38/wrky62 show the enhancing to pseudomonas syringae resistance (Kim et al., 2008).Closely A little years, some researches show that WRKY transcription factors works in the expression regulation of PR gene (PRs).ERF transcription because Son is called course of disease associated transcription factor, widely participates in the expression regulation of disease-resistant gene, what the albumen that it is encoded can be special In conjunction with the GCC-box original part in PR gene promoter, regulates and controls the expression of PR gene, played in disease resistance response Important regulating and controlling effect (B ü ttner and Singh et al., 1997；Dubouzet et al.,2003).

The immunologic mechanism induced along with the intracorporal signal network transmitting of plant, one of classical symptom is plant Hypersensitivity (HR) reaction can occur, cause the cell for infecting position and tissue necrosis, to prevent the diffusion of pathogen.Then, In in the period of certain, plant can generate resistance, referred to as systemic acquired resistance (SAR), while inducing plant body to pathogen A series of timely and effectively defense responses are generated, are mainly shown as the accumulation of cometabolism substance, the outburst of active oxygen, cell wall Modification and pathogenesis-related proteins (pathogenesis-related proteins, PR albumen) inducing expression etc., part Disease resistance response occur so that activation signal molecule excite entire plant immune response (Feys and Parker et al., 2000；Koornneef and Pieterse et al.,2008).Studies have shown that lipid molecule may be as class movement Signal causes SAR.DIR1 mutant obtains local disease resistance response, DIR1 and lipid transport egg after pathogen is handled White (LTPs) has sequence similarity, and may work (Maldonado et al., 2008) in signal transduction.Activity simultaneously Oxygen plays an important role in plant defense and SAR, and the expression of PR albumen is the mark of SAR.

Verticillium wilt pathogen (Verticillium dahlia) is a kind of fungal pathogens of soil-borne, can be infected more than 200 Kind dicotyledon, including the main industrial crops such as cotton, tomato, cucumber, capsicum, eggplant and potato, every annual meeting Cause huge economic loss (Fradin and Thomma et al., 2010；Inderbitzin and Subbarao et al.,2014).In nature, the life cycle of verticillium wilt pathogen be in the form of Microsclerotia by the root perceptual signal of plant simultaneously And sprout, the mycelia of sprouting penetrates the root surface of plant, (Vallad and Subbarao is then colonized in xylem vessel et al.,2008；Prieto et al.,2010；Zhao et al., 2014), once colonizing, fungi will be along vascular tissue Conidium and Microsclerotia are grown up and constantly generate, this will prevent the transport of moisture and eventually leads to the life of plant It is long it is slow, wilt or fallen leaves (Klimes et al., 2015).Due to Microsclerotia can with long-term surviving in plant residue and In soil, while the quantity of resistant gene in plant is very limited (Klosterman et al., 2015), and verticillium wilt pathogen is still very It is difficult to control, especially for some industrial crops, for example (,) cotton (Bejaranoalcazar et al., 1995；Wang et al.,2004).And plant resists the epidermal cell cell wall that the first line of defence that pathogen invades is exactly plant root, this is also Plant copes with one layer of mostly important defensive barrier of pathogen, will prevent most of pathogen.Since verticillium wilt is a kind of The disease infected by root, therefore particularly important effect is wherein just being played by the institutional framework resistance of pathogenic bacterium inducing, together When institutional framework variation can also activate the immunoregulation of plant entirety.

Suberin is a kind of Biopolvester of complex chemical composition, includes mainly some lipoid materials, is typically deposited on The cell wall of specific organization, such as root endodermis, exodermis, perithelium, kind skin and other edge tissues, form phellem layer. Phellem layer not only plays a significant role in control root system radial direction moisture and nutrient transport as a kind of protective barrier, And it can effectively resist the invasion (Franke and Schreiber et al., 2007) of pathogen.Potato tubers is created Wound can induce the suberification of perithelium, thus increase to the resistance of pathogen (Lulai and Corsini et al., 1998； Yossi et al.,2011).In addition, suberification can also improve soybean to the resistance of phytophthora, to avoid rotting for root and stem (Ranathunge et al.,2008).Inoculation phytophthora increases the suberification of soybean epiblem and endodermis.Further grind Study carefully, it was also found that the growth of mycelia is delayed by (Thomas et al., 2007) in the high kind of suberin content.Other one It has also been found that the intrusion of pathogen can induce suberification response (Southerton in a little plants, such as arabidopsis, wheat and tomato and Deverall et al.,1990；Franke et al.,2005；Botella et al.,2005).So cork mass-energy The intrusion of germ is blocked by the physical barrier of enhancing root or perithelial cells wall, but it is still necessary to the passes to control suberin synthesis Effect of the key gene in plant biological stress resistance is furtherd investigate.

At present, preliminary understanding there has been for the composition of suberification cell wall, but still for the structure of suberification polymer It knows little about it, chemical composition also needs further to identify.ω-hydroxy fatty acid of C16-C18 and α, ω-are bis- in the root of plant Carboxylic fatty acids (abbreviation α, ω-diacid) are the principal monomers for constituting suberin, are fatty acid in Cytochrome P450 ω-hydroxylation Formed under the catalysis of enzyme derivative (Werckreichhart and Feyereisen et al., 2000；Nawrath et al.,2002；Pollard et al.,2008).CYP86 subfamily in cytochrome P 450 monooxygenases family is mainly joined With the hydroxylating in the site fatty acyl-COA ω, ω-hydroxy fatty acid is formed, and a portion is oxidized to α, ω-diacid (Agrawal and Kolattukudy et al.,1978；Kurdyukov et al.,2006；Molina et al.)；This A little hydroxylated fatty acid finally transport cell wall by a series of polymerization reaction, have found during this complexity The participation of some reductases and transferase；Monomer finally needs transdermal delivery to cell wall simultaneously.At present for suberin list The mechanism of body transdermal delivery has just just begun one's study, wherein participating in the ATP-binding cassette of cutin and wax to cell wall transmembrane transport (ABC) transport protein and lipid transfer protein (LTPs) be the most key candidate albumen (Pighin et al., 2004； Choi et al.,2011；Deeken et al.,2016).In the biosynthetic process of suberin, CYP86 subfamily is played Vital effect.It is cloned into CYP86A1 for the first time in arabidopsis, expression discovery takes part in short chain rouge in yeast cells The hydroxylation (Benveniste et al., 1998) of fat acid.The root system C16 of the mutant cyp86A1 plant of arabidopsis and The suberin content of monomer of C18 is remarkably decreased, total aliphatic suberin content of monomer decline 60%, while passing through RT-PCR, GUS dyeing and cellular localization demonstrate CYP86A1 and are positioned in root endodermis endoplasmic reticulum, this also reflects suberin monomer Synthesis carried out on the net in endoplasm (Et al., 2008).By the homologous gene CYP86A33 silencing of CYP86A1 in potato Afterwards, perithelium suberin content is substantially reduced (Serra etal., 2009).The CYP86A2 in arabidopsis, CYP86A4 and CYP86A8 is three closer genes of affinity, works in the biosynthetic process of extracellular lipid, also joins With the hydroxylating of short chain fatty acids, but at present most research results all indicate that these three genes have participated in cell wall cutin In forming process, and the related fatty acids of CYP86A2 participation synthesis may inhibit the table of type III gene in pathogen Up to (Xiao et al., 2004；Libeissonet al.,2009；Wellesen et al.,2001).And CYP86B1 with CYP86A1 belongs to the inhomogeneity of a subtribe, as a kind of long chain fatty acids hydroxylase, participates in C22-C24 long chain fatty acids Hydroxylating (Compagnonet al.,2009).In arabidopsis the root of cyp86B1 mutant and kind skin in C22 and ω-the carboxylic acid and α of C24, ω-diacid almost lack, but suberin monomer C22 and C24 fatty acid cumulative rises (Molina et al.,2009)。

Seldom, there has been no the correlations of CYP86 family gene for research relevant for suberin biosynthesis in cotton at present Report.Cotton is the important sources of cotton fiber and cottonseed oil as one of industrial crops important in the world.But cotton yellow The evil of being critically ill of withering is huge, has a very wide distribution since its disease fungus has, and harm is serious, and host range is wide, spread speed it is fast and The time-to-live waits so long feature in the soil, be difficult to administer, the serious yield and quality for affecting cotton, referred to as cotton " Cancer ".Therefore the intrusion success rate of pathogen is avoided to be only key as far as possible from picking up at all the prevention and treatment of verticillium wilt pathogen. Currently, for the pathogenic mechanism of verticillium wilt pathogen, opinions vary, and cotton and the mechanism of verticillium wilt pathogen interaction are still unclear, plant roots The cell wall tissue in portion is the important barrier for defending pathogen invasion, and it is related to carry out some cell wall polyester synthesis in cotton The research of gene has important directive function for the prevention and treatment of verticillium wilt pathogen.

Summary of the invention

It is new in raising Cotton disease resistance and cultivation cotton that the object of the present invention is to provide a kind of cotton GbCYP86A1-1 genes Application in germplasm.The gene, which is demonstrated, using the instantaneous silent technology of gene and transgenic arabidopsis is related to cotton and arabidopsis To the resistance of verticillium wilt.Using this gene as target gene, pass through the gene engineering methods such as transgenosis, overexpression GbCYP86A1-1 Gene, cultivates that growth and development is good, and production of cotton fibers and quality are normal, and the cotton new germ plasm that significantly improves of resistance and in life It is applied in production.

Another object of the present invention is to provide a kind of methods for improving verticillium wilt resistance of cotton by same.

Another object of the present invention is to provide a kind of cotton GbCYP86A1-1 gene, and the gene is provided in sea island cotton H7124 A subgroup (GbCYP86A1-1A) and genome and full-length cDNA ORF nucleotide in D subgroup (GbCYP86A1-1D) Sequence.The amino acid sequence that the gene encodes in A subgroup and D subgroup in sea island cotton.

The purpose of the present invention is achieved through the following technical solutions:

The GbCYP86A1-1 gene as shown in SEQ ID NO.1 or SEQ ID NO.2 is improving cotton and model plant Arabidopsis cultivates the application in cotton new germ plasm to resistance to verticillium wilt.

Above-mentioned application, is: being improved using the overexpression of the GbCYP86A1-1 gene into arabidopsis quasi- The disease resistance of southern mustard.Using the GbCYP86A1-1 gene as target gene, by gene engineering methods such as overexpression technologies, It is normal to cultivate growth and development for overexpression GbCYP86A1-1 gene in cotton, and the cotton that resistance to verticillium wilt significantly improves is new Germplasm is simultaneously applied in production.

A method of improve verticillium wilt resistance of cotton by same, in cotton overexpression nucleotide sequence such as SEQ ID NO.1 or GbCYP86A1-1 gene shown in SEQ ID NO.2.

One can significantly improve the GbCYP86A1-1 gene of cotton and arabidopsis disease resistance, which is (1)~(3) In any one:

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

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

(3) other cotton seeds are derived from, are higher than 98% and function phase with the nucleotide sequence homology of (1) or (2) described gene Same or similar nucleotide sequence.

The albumen encoded by GbCYP86A1-1 gene, the gene GbCYP86A1-1A as described in (1), gene coding Albumen has the amino acid sequence as shown in SEQ ID NO.3；Gene GbCYP86A1-1D as described in (2), gene coding Albumen have the amino acid sequence as shown in SEQ ID NO.4.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned GbCYP86A1-1 gene.

Above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium is improving Cotton disease resistance and is cultivating cotton Application in new germ plasm.

The disease resistance is verticillium wilt disease resistance.The new germ plasm is the cotton novel species that resistance to verticillium wilt significantly improves Matter.

Advantages of the present invention is shown:

(1) structure and functional analysis for carrying out system for the CYP86 subfamily gene in cotton for the first time, identify wood Important function of the bolt matter synthesis related gene GbCYP86A1-1 in cotton verticillium wilt resistance.

Up to the present, it is ground in cotton there are no the correlation about fatty acid ω-hydroxylase CYP86 gene family member Study carefully report.We are reference with the genome of cotton, have carried out detailed point to all CYP86 subfamily members in cotton Analysis carries out network analysis to family gene by structure, evolution and expression.CYP86A in cotton there are three homologous gene, It is respectively distributed on different chromosome, and function is broken up, and is had found wherein using molecular biology experiment Important function of the GbCYP86A1-1 in the formation of cell wall fatty acid biological polyester and verticillium wilt induction immune resistance, this And first passage grinds CYP86 subfamily member in the Cytochrome P450 superfamily in cotton compared with the method for system Study carefully, provides reference and new gene for the research of root Biopolvester in cotton and cotton disease resistance molecular breeding.

(2) transgenic arabidopsis of GbCYP86A1-1 overexpression be study gene caused by disease resistance variation with And transgenosis can be enhanced in the elite clone of the resistance signal's access influenced and molecular mechanism, overexpression GbCYP86A1-1 The disease resistance of Arabidopsis plant.

Arabidopsis can be enhanced to the Disease Resistance Identification discovery overexpression GbCYP86A1-1 of transgenic arabidopsis to wither to Huang The resistance of disease, the root of transgenic plant is more difficult to be invaded by pathogen compared with wild type material, the verticillium wilt pathogen in each tissue Biomass is less, and disease index is lower.And more relevant time of Biopolvester synthesis is not only had found in transgenic plant The variation of grade metabolic process and participation lipid are shifted to ATP-binding cassette (ABC) transport protein and lipid of cell wall transmembrane transport The variation of albumen (LTPs) even more therefrom has found the enhancing of disease-resistant related immune access, and which includes receptoroid albumen to swash The change of enzyme and receptoroid albumen, ERF course of disease associated transcription factor and WRKY transcription factor and a large amount of PR pathogenesis-related proteins Change, the expression of these genes all shows significantly to increase.From result, we can be found that transgenic plant not only enhances root Physical structure resistance, and enhance some disease-resistant accesses of induction excited by signaling molecule, this is also some participation secondary The research direction of the pathway gene of metabolism provides new thinking and reference, while GbCYP86A1-1 gene regulation disease resistance It was found that also laying the foundation further to excavate cotton disease resistance gene and cultivating disease-resistant variety.

Detailed description of the invention

The network analysis of Fig. 1 diploid Lei Mengdeshi cotton GrCYP86 gene family member

The evolutionary analysis of a:GrCYP86 gene family member.The tetraploid rice of b:GrCYP86 gene family member.C: The genomic organization of GrCYP86 gene family member.D:GrCYP86 gene family member's structural domain.

The expression analysis of Fig. 2 GhCYP86 gene family member

Spatial expression pattern analysis of the a:GhCYP86 gene family member in different tissues organ.B: special table in root The GhCYP86B1-1 of predominant expression in three genes GhCYP86A1-1, GhCYP86A1-2, the GhCYP86A1-3 and root reached The expression variation of different time under verticillium wilt pathogen induction.Using disease-resistant material H7124 and susceptible material army cotton No. 1 as it is anti-, feel Material control.

The verifying of Fig. 3 VIGS gene silencing system

A: the sea island cotton of silencing CLA1 gene shows the phenotype of blade albefaction.B: disease-resistant material H7124 with susceptible material No. 1 verticillium wilt pathogen V991 processing of army cotton is compared with the phenotype of water process.C:VIGS individually silencing GbCYP86A1-1, GbCYP86A1-2, GbCYP86A1-3 influence the expression of other two homologous gene.

Fig. 4 silencing GbCYP86A1-1, GbCYP86A1-2, GbCYP86A1-3 plant susceptibility in sea island cotton improves

A: individually silencing GbCYP86A1-1, GbCYP86A1-2, GbCYP86A1-3 and common heavy in sea island cotton After silent three genes, the quantitative PCR of gene expression dose is verified.Verticillium wilt pathogen is inoculated with after b:VIGS cryptiogene in different time Susceptible phenotype.C: the plant disease grade investigation after silencing different genes.D: the upward 1cm plant stalk of cotyledon knot is split after connecing disease 15 days Bar phenotype.

Chromosome location, subcellular localization and its different plant species where tri- homologous genes of Fig. 5 GbCYP86A1 is homologous The phylogenetic analysis of gene

Position of the a:GbCYP86 family homologous gene on Lei Mengdeshi cotton colour solid.B: different plant species source CYP86A1 Phylogenetic analysis and tetraploid rice.The subcellular localization of tri- genes of c:GbCYP86A1.

The Disease Resistance Identification of tri- gene transgenic arabidopsis of Fig. 6 GbCYP86A

A: the Molecular Detection of transgenic arabidopsis.B: different carriers connect the phenotype of transgenic arabidopsis after disease 2 weeks.C: different Carrier disease grade statistics and sick grade index analysis.D: different carriers connect root after disease 10d, stem, in leaf verticillium wilt pathogen relative amount. E: stem section at the upward 1cm of different carriers lotus throne leaf, germ recovery analysis after potato culture medium culture 3 days.

Tri- gene transgenic Arabidopsis plant phenotypic characteristics of Fig. 7 GbCYP86A.

4 weeks transgenic arabidopsis are grown in native basin compared with wildtype Arabidopsis thaliana growth and development, phenotype becomes without obvious Change.

The microexamination of the tri- gene transgenic arabidopsis seedling stage roots Fig. 8 GbCYP86A.

A: the phenotype of root long.B: root cell length.C: root hair phenotype.D: the statistics of root long.E: root cell length system Meter.F: root staple length statistics.

Fig. 9 GbCYP86A1-1 transgenic arabidopsis root transcript profile GO enrichment analysis, tri- genes of GbCYP86A turn base Histotomy microexamination and the detection of C16-C18 fatty acid relative amount because of arabidopsis maturity period root.

A: comparing the root transcriptome differences of GbCYP86A1-1 transgenic arabidopsis and wildtype Arabidopsis thaliana, GO enrichment Biological processes.B: taking maturity period arabidopsis main root, apart from ground base portion 2cm sample, carries out histotomy.C: samples taken production Ultra-thin section, at middle part, cutting thickness is 2 μm of progress lipid dyeing observations.D: the relative amount of root fatty acid is surveyed It is fixed.

The difference expression gene identified is sequenced with root transcript profile is compareed in Figure 10 GbCYP86A1-1 transgenic arabidopsis

On the basis of statistical difference, difference expression gene of the FDR less than 0.05 is extracted, wherein more than 1.5 times of differences Gene is roughly divided into two classes, participates in cometabolism process and anti pathologic immunity process respectively.

Figure 11 GbCYP86A1-1 transgenic arabidopsis verticillium wilt pathogen handles 3 days back root part transcript profile sequencing analysis and root The free-hand section in portion

A:GbCYP86A1-1 transgenic arabidopsis is handled respectively with verticillium wilt pathogen laggard with water process with wildtype Arabidopsis thaliana Row differential gene number compares, and carries out GO enrichment respectively.B: verticillium wilt pathogen carries out free-hand section, observation to root after infecting 3d Pathogen invades situation.

The quantitative PCR of Figure 12 transcriptome analysis result is verified

A: 30 genes for randomly selecting 1.5 times of transcript profile sequencing result fold differences or more after water process are quantified The verifying of PCR draws scatter plot and calculates R²Value examines degree of fitting.Abscissa is GbCYP86A1-1/ in transcript profile sequencing result The fold differences of WTBase value, as a result take log2；Ordinate is quantitative PCR result 2^-△Ct, as a result take log2.B: ibid 30 bases Because of the quantitative PCR verifying after verticillium wilt pathogen V991 processing.

Cometabolism process and anti pathologic immunity are participated in Figure 13 GbCYP86A1-1 transgenic arabidopsis and wildtype Arabidopsis thaliana The quantitative PCR detection of process related gene

A: the quantitative PCR verifying of cometabolism related gene is participated in.B: receptor albuminoid kinases (RLKs) and receptor class The quantitative PCR of albumen (RLPs) is verified.C: the quantitative PCR verifying of associated transcription factor (ERFs, WRKYs).D: course of disease correlation egg The quantitative PCR of white gene (PRs) is verified.

Figure 14 in cotton silencing GbCYP86A1-1 gene pairs PR gene expression influence

A: the expression variation of 8 PR genes different time under verticillium wilt pathogen induction in sea island cotton.B: heavy PR gene PR1, PR5 extremely significant downward expression compared with the control after silent GbCYP86A1-1 gene, PR2, PR4, PR16 significantly lowers expression.

The cell wall structure resistance and Physiology and biochemistry resistance of Figure 15 GbCYP86A1-1 gene influence plant

GbCYP86A1-1 gene can promote the bolt matter of cell wall after being induced by verticillium wilt pathogen, plant is made to generate induction Structural resistance；And the variation of lipid and the modification of cell wall will affect Physiology and biochemistry resistance, the immune response in active cell.

Specific embodiment

The method that the present invention utilizes bioinformatics, identifies from the genome of the diploid Lei Mengdeshi cotton discharged 10 CYP86 family genes, and its homologous gene further is excavated out from the upland cotton TM-1 and sea island cotton 3-79 being sequenced, Hierarchial-cluster analysis is classified as A, B, C three classes.Histoorgan expression analysis finds that the CYP86A1 genoid in cotton is being planted Specifically expressing in the root of strain.Wherein CYP86A1-1 gene expression dose is significantly associated with disease resistance.With susceptible upland cotton army cotton 1 It number compares, in disease-resistant sea island cotton H7124 root tissue, expression and induced expression level significantly increase the gene.Based on PCR Technology obtains the genome sequence and overall length ORF sequence of CYP86A1-1 in sea island cotton H7124, and the gene is in allotetraploid The nucleotide sequence of genome and full-length cDNA ORF in the genome A subgroup (GbCYP86A1-1A) of sea island cotton H7124 is such as Shown in SEQ ID NO.1；Genome of the gene in the genome D subgroup (GbCYP86A1-1D) of sea island cotton H7124 and complete The nucleotide sequence of long cDNA ORF is as shown in SEQ ID NO.2.The gene is free of introne, gene coding region genome sequence It arranges identical with overall length ORF sequence.By verticillium wilt pathogen Resistance Identification, it is based on VIGS technology silencing base in sea island cotton H7124 Because significantly reducing the resistance to verticillium wilt of cotton, by the gene, overexpression significantly improves disease resistance in arabidopsis.

(1) phylogenetic analysis of CYP86 gene family, structural analysis and expression pattern analysis in cotton

All CYP86 family genes are searched in the full-length genome of diploid Lei Mengdeshi cotton, according to software Cytochrome P450 (PF00067) domain protein family is extracted in cotton and owns in the website HMMER3.0 and Pfam P450 superfamily gene, and with SMART and INTERPROSCAN website authentication these prediction family gene.According to 11 CYP86 family genes carry out clustering in arabidopsis, share 10 CYP86 family genes (table 1) in cotton.

Using the name of the CYP86 gene family in arabidopsis as reference, according to the homology with it to CYP86 in cotton Family gene has carried out the name of system, and CYP86A class has 7 genes in Lei Mengdeshi cotton, and CYP86B class has 2 genes, CYP86C class has 1 gene (Figure 1A).Wherein GrCYP86A1 is there are three homologous gene, be respectively designated as GrCYP86A1-1, GrCYP86A1-2,GrCYP86A1-3；GrCYP86A7 there are two homologous gene, name respectively GrCYP86A7-1, GrCYP86A7-2；There are two homologous genes by GrCYP86A8, name GrCYP86A8-1, GrCYP86A8-2 respectively；And Also there are two the CYP86B1 in gene and arabidopsis to cluster to a branch by GrCYP86B1, but homology is not high, temporarily orders Entitled GrCYP86B1-1, GrCYP86B1-2 (Figure 1B).By website (GSDS) 2.0 (http: // Gsds.cbi.pku.edu.cn/) and corresponding genome sequence encodes CDS sequence with it and carries out to the structure of family gene Analysis, wherein only 3 genes have introne (Fig. 1 C)；Prediction using the website SMART to structural domain removes GrCYP86A7- All there is P450 Core domain and transmembrane domain (Fig. 1 D) other than 1 and GrCYP86A7-2.In short, according to clustering and CYP86 family gene in cotton has been carried out the classification and name of system to homology analysis by we, analyzes gene structure And Domain, it has been found that the structure of CYP86 family gene is relatively conservative.

In order to study effect of the CYP86 family gene in tetraploid cotton, according to upland cotton G.hirsutumacc.TM- 1 transcript profile data, analyze gene at nutrition organs (root, stem, leaf), floral organ (petal, stamen, gynoecium), ovule (- 3, 0,3 days) and fibr tissue (5,10,20,25 days) expression pattern, FPKM value takes log2.As a result, it has been found that wherein GhCYP86A1 class Three homologous gene specifically expressings in root；And GhCYP86A8 class is mainly expressed in floral organ, ovule and fiber； GhCYP86A7 class is mainly expressed in stem, leaf, petal and 10 days fibers；GhCYP86B1-1 shows as constitutive expression mould Formula, expresses all higher in each tissue, and GhCYP86B1-2 has only detected A subtribe in tetraploid and only in floral organ It official and is expressed in fiber for 25 days；GhCYP86C1 is not expressed substantially in each tissue.CYP86 family gene has guarantor in cotton Still function but has diversity to the gene structure kept, and spatially different expression patterns may be with it in the intracorporal function of plant (Fig. 2A) can be associated.

The function of the CYP86 family related gene of existing species report, all focuses on the biosynthesis of lipid polyester substantially, Effect mainly prevents the missing of moisture, prevents extraneous biology and abiotic stress from influencing, enhances the resistance of plant.In order to Prove that CYP86 family gene acts in resistance mechanism, we are with the root of verticillium wilt pathogen High pathogenicity bacterial strain V991 inducing cotton Portion, according to three homologous genes and CYP86B1-1 for expressing high CYP86A1 class in transcription group selection root, when different Between have detected the variation of expression, respectively using disease-resistant material sea island cotton H7124 and susceptible material upland cotton army cotton No. 1 as Anti-, sense control.Wherein, four genes all show have higher expression in disease-resistant material sea island cotton H7124, and Three homologous genes of CYP86A1 class all show significant up-regulated expression in different materials and the different time sections of induction Trend induces 144 hours later periods expression quantity to improve several times (Fig. 2 B) especially in sea island cotton.The GbCYP86A class in sea island cotton Three homologous genes be likely to perception verticillium wilt pathogen intrusion have activated corresponding anti pathologic immunity process, to prevent pathogen Further colonize.Research the primer is listed in table 2

Table 2: special primer used in inducing expression

Primer	Primer sequence (5'-3')	Purposes
			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	Cotton quantifies internal standard
Y8991R	AATTTCACGAACAAGCCTCTGGAA	Cotton quantifies internal standard

(2) VIGS technology Preliminary Identification GbCYP86A1 genoid participates in Cotton disease resistance

As described above, GbCYP86A1 genoid may important role during verticillium wilt resistance of cotton by same.In order into one Effect of the step research GbCYP86A1 genoid in resistance mechanism, we are distinguished in sea island cotton special by VIGS technology One in three genes of silencing GbCYP861A class, and chooses 3 genes of homologous fragment while silencing and carry out the preliminary of functions Identification.Have chosen the special segment in the 3 ' areas UTR respectively, construct TRV2:GbCYP86A1-1, TRV2:GbCYP86A1-2, Tetra- carriers of TRV2:GbCYP86A1-3 and TRV2:H3091 are with the endogenous gene in silencing H7124, and wherein TRV1-TRV2 makees To compare simulation process.In order to verify the feasibility of VIGS system in cotton, coding 1-deoxy-D-xylulose -5- phosphorus is used The CLA1 gene of acid synthase as reference, after silencing the blade and stem of plant have albefaction phenotype (Gao et al., 2013).All carriers built are injected into the cotyledon of cotton seedling by the method for mediated by agriculture bacillus, seedling about seven days Size, the seedling that do not inject are also cultivated in identical environment as control.All TRV2-CLA1 carriers of having injected after two weeks All there is the consistent albefaction phenotype of high uniformity (Fig. 3 A) in cotton leaf.In order to further determine the silence efficiency of gene, respectively A silencing strain and control strain extract RNA respectively, and the expression of gene is verified by real-time fluorescence quantitative PCR (qPCR).With Do not inject control H7124 and inject TRV2 empty carrier compared to silencing strain GbCYP86A1-1, GbCYP86A1-2, The expression of GbCYP86A1-3 and H3091 is lower, shows that these genes are all extremely significant on transcriptional level and is silenced, and special The expression for changing other two homologous genes of the one of gene pairs of silencing influences simultaneously less (P < 0.01) (Fig. 3 C).

After determining target gene silencing, Disease Resistance Identification is carried out in one heart stage of two leaves.With the verticillium wilt pathogen V991 of culture, 25mL 1×10⁷Suspension spore liquid, tear bottom and hurt the mode of root and carry out connecing disease.Each material plants 24 plants, and 3 repetitions are arranged. It connects in three days after being ill and does not water, rear every two days moisturizing 50ml.Guarantee indoor temperature and humidity.Wherein resistant variety H7124 with And susceptible variety army cotton No. 1 is respectively intended to as control, after connecing bacterium 10 days, the cotyledon that army cotton is No. 1 shows significantly to wither, and H7124 just shows the phenotype that a small amount of blade turns yellow when 15 days.Table after inoculation verticillium wilt pathogen V99120 days and 25 days Type shows (Fig. 3 B).After connecing disease about 3 weeks, the cotton seedling of silencing plant, especially TRV2:GbCYP86A1-1, TRV2: GbCYP86A1-2 and TRV2:H3091 blade shows more wilting and yellow, and observes 25 days and 30 days, equally Three silencing material disease incidence are higher, and wherein the susceptible phenotype of TRV2:GbCYP86A1-1 and TRV2:H3091 is more serious (such as Fig. 4 B)；The army cotton equally handled No. 1 when 25 days blade just fallen ill and fallen off substantially.Cryptiogene in a word 3 genes of GbCYP86A1-1 and simultaneously silencing can dramatically increase the verticillium wilt sensibility of H7124, and silencing GbCYP86A1-2 can also improve sensibility to a certain extent, but silencing GbCYP86A1-3 performance is not it is obvious that display Different homologous gene function differentiations react different to disease resistance.Furthermore our each processing have selected the sick grade investigation of 24 plants of progress, As a result only the blade of seldom morbidity quantity has been arrived in investigation in the TRV2:00 plant of control and the H7124 not injected, 35 It when disease leaf rate be about 55%.However the H3091 plant of cryptiogene GbCYP86A1-1 and coprecipitated silent 3 genes Sick leaf rate has all reached 86%, and the sick leaf rate of the plant of silencing GbCYP86A1-2 and GbCYP86A1-3 gene is respectively 77% and 69%, the sick leaf rate of susceptible material army cotton has had reached 100% (such as Fig. 4 C).

Verticillium wilt pathogen is soil-borne fungus, and the root intrusion through plant spreads all over whole body by vascular tissue.Verticillium wilt pathogen infects Afterwards, plant vasular tissue can be blocked, makes it that brownish black (Fradin and Thomma et al., 2010) be presented.The journey infected It is deeper to spend higher color, content of the verticillium wilt pathogen in vascular tissue can intuitively be shown by splitting bar experiment.Connecing disease 15 days After, beveling is carried out with the same area (the upward 1cm of cotyledonary node) of silencing plant and adjoining tree and splits bar experiment, and in body It takes pictures under formula mirror observation.Similarly it is observed that the plant of three genes of silencing GbCYP86A1-1 and simultaneously silencing ties up pipe Tissue has invaded more verticillium wilt pathogens, the followed by plant of silencing GbCYP86A1-2 and GbCYP86A1-3, and adjoining tree And H7124 only has minimal amount of mycelia intrusion, there is no any mycelia (Fig. 4 D) for the conduct control of water process.

By comparing the plant of each gene of silencing and the incidence of control group plant, GbCYP86A1 genoid is in difference Plant is imparted in degree for the resistance (P < 0.01) of verticillium wilt pathogen.Wherein, GbCYP86A1-1 is that cotton resists verticillium wilt pathogen Important gene, compared to other homologous gene phenotypes it is more significant, had potential application in cotton disease resistance breeding.Research The primer is listed in table 3

Table 3: amplification the primer

(3) chromosome location of GbCYP86A1 genoid, evolutionary analysis and positioning

GbCYP86 family gene is all distributed on different chromosome substantially in cotton tetraploid, wherein GbCYP86A1 3 homologous genes of class are distributed on three chromosomes.On No. 3 chromosomes, GbCYP86A1-2 contaminates GbCYP86A1-1 at No. 7 On colour solid, and GbCYP86A1-3 is on No. 5 chromosomes.We obtain other more species in ncbi database again simultaneously The amino acid sequence of middle CYP86A1 gene is respectively derived from sea island cotton, upland cotton, Lei Mengdeshi cotton, arabidopsis, cocoa, poplar The plants such as tree, grape, tobacco, sweet wormwood, sunflower, soybean, red bean, rice, corn.It can be seen that GbCYP86A1 is different It is widely present in species.And the drafting of systematic evolution tree is carried out by the comparison between amino acid sequence, analysis is soft using MEGA5 Part program, parameter used is default setting, while application software DNAMAN compares homology.The result shows that each cotton variety In CYP86A1 homology it is very high, it is close compared with the affinity of cocoa and poplar.GFP4 subcellular localization carrier is constructed, Using the method for Agrobacterium injection tobacco leaf, the subcellular of confocal laser scanning microscope GbCYP86A1 genoid is utilized Positioning, 3 genes are all located in endoplasmic reticulum and coupled membranous system (figure as most P450 family gene 5).Subcellular localization carrier is listed in table 4.

Table 4: subcellular localization vector construction is used to recombinate primer

(4) GbCYP86A1 genoid Disease Resistance Identification and phenotypic analysis after overexpression in arabidopsis

Furtherd investigate in order to which the function to GbCYP86A1 genoid is further, we by GbCYP86A1-1, On the GbCYP86A1-2 and GbCYP86A1-3 tri- gene constructed over-express vector PBI121 started to 35s, pass through agriculture bar The method that bacterium mediates is transformed into arabidopsis wild type (Col-0), the screening positive clone on resistance Selective agar medium, and is mentioned The DNA of positive plant is taken to be identified, after screening T3 generation by generation, our final each identified for genes 6 homozygous to turn base Because strain is cloned, it is respectively designated as OE1-OE6.Us, which are cloned, for 6 of each transgenic line is extracted the RNA of root, Have detected the overexpression situation of foreign gene, Integrative expression feature, we have chosen respectively the highest two clone OE1 of expression with OE2 is further analyzed (Fig. 6 A).

First to each transgenic line two clone phenotype observed, when four true leaves of arabidopsis from Culture medium moves on to Tu Penzhong, and discovery phenotype has no significant change (Fig. 7) after cultivating four weeks.Then verticillium wilt is inoculated with using root dipping method Bacterium V991, suspension spore liquid concentration are 1 × 10⁷Spores/ml observes incidence after two weeks, and counts morbidity series, Disease index is calculated, while two weeks are grown with the wild type WT that back root part dips in water normally, as control (Fig. 6 B).As a result wild type Disease index of the arabidopsis WT after connecing disease two weeks has reached 72.5%, and the quasi- south of overexpression GbCYP86A1-1 gene Two clone's diseases of mustard refer to only 30% and 14%, and resistance to verticillium wilt is remarkably reinforced；While overexpression GbCYP86A1-2 gene Two clone's diseases of arabidopsis refer to there is 42% and 55%, and resistance also increases；The arabidopsis of overexpression GbCYP86A1-3 gene Two clone's diseases, which refer to, has reached 66%, and compared with wild type, apparent variation (Fig. 6 C) is not detected in resistance.It withers to detect Huang The intrusion situation of germ, we have taken each transgenic line difference to clone and compare strain WT's respectively after connecing disease 10 days The identical position of root, stem and leaf, has detected the relative amount of verticillium wilt pathogen in tissue respectively, and each tissue is ground to powder and is extracted Genomic DNA, special using the specific primer I TS-F and verticillium wilt pathogen of the DNA transcribed spacer based on ribosome internal Reverse primer ST-Ve1-R measures the content of fungi, using cotton internal standard gene as control.The result shows that after inoculation 10 days, mistake Transgenic arabidopsis two of amount expression GbCYP86A1-1 gene, which are cloned in, has only detected seldom verticillium wilt pathogen in root, stem and leaf Content, especially in blade.GbCYP86A1-2 be overexpressed plant respectively organize in verticillium wilt pathogen relative amount also compared with It is low, and GbCYP86A1-3 be overexpressed plant and wild type WT respectively organize in detect very high relative amount (Fig. 6 D).Knot Fruit analysis shows, GbCYP86A1-1 and GbCYP86A1-2 are overexpressed the initial stage that strain infects and just show resistance, only very Least a portion of disease fungus invades root and colonizes in vascular bundle.We further devise the Huang infected in initial stage intrusion stem Wither germ recovery experiment, analyze the infection rate of arabidopsis.Each transgenic line randomly selects 5 after connecing disease 7 days, carries out normal Tissue separation is advised, takes the stem section of 2cm upwards from a kind of sedge root section, with 70% alcohol surface sterilization, aseptic water washing 3-4 times, Every be placed in PDA culture medium, 25 DEG C after constant temperature incubation 4 days, after verticillium wilt pathogen bacterium colony is grown, are observed and count clump count Amount.For the WT of water process as control, there is no bacterium colonies to grow.Two grams of the transgenic plant of overexpression GbCYP86A1-3 Grand and wild type WT connects the later stipes of disease and obviously restored more bacterium colonies, and overexpression GbCYP86A1-1 turns base Because plant has only restored especially few bacterium colony (Fig. 6 E).

It is comprehensive these as a result, we have enough cards it was demonstrated that compared with GbCYP86A1-2 and GbCYP86A1-3, GbCYP86A1-1 gene can more obviously enhance arabidopsis to the resistance of verticillium wilt pathogen, and disease resistance be mainly manifested in for Verticillium wilt pathogen invades in the resistance at initial stage, and the intrusion of more verticillium wilt pathogens can be kept out at the initial stage of infecting, makes least a portion of cause of disease Bacterium colonizes in vascular tissue, this direction also studied for plant disease-resistant provides more thinkings.Research the primer is listed in table 5

Table 5: over-express vector constructs digestion primer and quantitative detection primer

(5) structure observation and GbCYP86A1-1 of GbCYP86A1 genoid overexpression back root part in arabidopsis The transcriptome analysis of transgenic arabidopsis root

We have chosen in each transgenic line two clones, and the more obvious clone of phenotype carries out subsequent grind Study carefully, wherein GbCYP86A1-1 transgenic line has chosen OE2, GbCYP86A1-2 and GbCYP86A1-3 and has chosen OE1 respectively, Overexpression number OEs is omitted in subsequent figures.

In order to deeper into research GbCYP86A1-1 function, we hang down to the different strains of different transgenosis Straight culture, has investigated the seedling root long in culture basal growth 8 days；It is contaminated simultaneously with the Propidium Iodide (PI) of 50 μ g/mL Color 30s carries out the observation and measurement of cell length in fluorescence microscope；The case where using body formula sem observation and counting root hair, often 5 repetitions of a material, each duplication at least ten root staple length.Survey data is integrated, by statistical analysis, discovery is not With its root long between transgenic line and control, root cells length and root staple length there are no significant difference.Show 3 transgenosis Growth (Fig. 8) of the arabidopsis strain without influence on Arabidopsis thaliana Seedlings phase root.

Arabidopsis due to turning GbCYP86A1-1 gene shows extra high resistance to verticillium wilt pathogen, in order to complete Its function is preferably parsed in genomic level, we divide 4 weeks big wild type WT and GbCYP86A1-1 transgenic arabidopsis Root RNA Yong not be extracted, transcriptome differences expression analysis is carried out after water process and verticillium wilt pathogen V991 handle three days.

Analysis is compared to two materials of water process first, according to sequencing result, differential expression genes is screened, chooses Gene of the fold differences greater than 1.5 times carries out GO enrichment.As the result is shown in GbCYP86A1-1 transgenic arabidopsis root with stress, The related genes such as biostimulation, defense response, plant immune, redox, cometabolism and cell wall structure are by significant rich Collect (Fig. 9 A).

For the structural resistance relevant entry of transcript profile enrichment, including cometabolism, lipid transport, phenylpropyl alcohol alkane metabolic process In flavonoids synthesis and cell wall external structure, GbCYP86A1-1 gene may participate in changing the lipid of arabidopsis The structure of metabolism and cell wall is to achieve the purpose that resist verticillium wilt pathogen.So we were to 4 weeks big maturity period arabidopsis Root carried out the microexamination in cross section, base portion 20mm apart from main root samples 10mm (Fig. 9 B), carries out semithin section Production, by the embedding of fixation, dehydration and resin, is fixed on slicer for sample, is cut into cutting for 2.0 μ m-thicks in middle position Piece, with lipophilic dyes the Sudan 7B (0.1%, w/v；In 50% (v/v) PEG400,45% (v/v) glycerol, in 5% water) to thin Born of the same parents dye, and room temperature 1 hour, after dyeing, are rinsed with SDS (1%), then wash with water and be placed on glycerol: micro- in water (1:1) Observation.As a result, it has been found that the root okioplast cell wall of GbCYP86A1-1 transgenic arabidopsis occurs more compared with wild type WT More Ester accumulations, red is deeper to be become apparent from, and the also densification more compact of the structure of cell, it may be possible to improve disease resistance Key reason (Fig. 9 C).We further have detected the fatty acid relative amount in different transgenic line roots, configure first Various concentration external standard draws standard curve, then grinds sample sufficiently, carries out corresponding depolymerization reaction, esterification processing, with 10 μ g dotriacontanes carry out the measurement of relative amount using gas chromatograph as internal standard.As a result turn base in GbCYP86A1-1 It is higher than other strains (Fig. 9 D) because the relative amount of C16-C18 fatty acid in the root of arabidopsis is extremely significant.Synthesis result analysis GbCYP86A1-1 gene affects the fatty acid metabolism of arabidopsis root cell and the lipid content of cork confluent monolayer cells, thus One of structural barriers is formed in root, keeps out the disease fungus intrusion of the overwhelming majority.

The biological function that CYP86A1 was once studied in arabidopsis is mainly joined as a kind of hydroxylase positioned at endoplasmic reticulum With the hydroxylating of fatty acid, hydroxylated fatty acid can by a series of redox reaction and fatty acid chain extension most Final polymerization requires that cytoplasma membrane is exported and passed through from endoplasmic reticulum, then gathers at complicated Biopolvester, these polymer Cell wall forms suberin.Some researches show that lipid transfer proteins (LTPs) and ATP-binding cassette (ABC) to transport egg at present The transdermal delivery (Vishwanath et al., 2015) of white ginseng and suberin component.By transcriptome analysis, we are enriched to Some LTP transport proteins participate in transmembrane transport, while being also enriched to a large amount of abc transport albumen and AAA-ATPase, all It is to work in the supply of energy with ATPase active gene.These genes are all shown significantly in transgenic plant High expression, since the overexpression of GbCYP86A1-1 gene promotes the intracorporal lipid aggregate of plant and transhipment, need LTP transport protein supports transmembrane transport and abc transport albumen to provide required energy.It has also been enriched to many participations time simultaneously The gene of grade metabolism process, these genes take part in phenylpropyl alcohol alkane metabolism process or suberin synthesis process, in transgenic line Middle expression also significantly improves.Such as phenylalanine lyase (PAL) is in catalysis phenylalanine metabolic pathway step 1 reaction process Enzyme and the process rate-limiting enzyme, 1 amino-cyclopropane -1- carboxylic acid (ACC) oxidizing ferment and PAL are in the agglutination of damage All play an important role (Kato et al., 2000).KCS gene encodes 'beta '-ketoester CoA synzyme (KCS), is fatty acid Key enzyme (Joubes et al., 2000) during extension.Show P450 wide participation to phenylpropyl alcohol alkane metabolic process and secondary The synthesis process of grade metabolite provides the candidate gene of reference for the research of suberin biosynthetic process.

The possibility regulatory factor of suberin synthesis is located at WRKY-, NAC-, MYB- specific transcription factor region, these genes The priority expression (Kilian et al., 2000) in the tissue of suberification.Course of disease correlation (ethylene responses) (AP2/ERF) transcription The factor participates in secondary cell wall modification (Lasserre et al., 2000).But these transcription factors participate in suberification process tune The positive evidence of control is also insufficient, needs further in-depth study.It has been found that many WRKY turns in transcript profile Record the factor and ERF transcription, at the same be also found many Receptor-like protein ki-nases (RLKs), receptoroid albumen (RLPs) with And pathogenesis-related proteins (PRs), and high expression is all shown in GbCYP86A1-1 transgenic line, thus it is speculated that may be by The induction for affecting some signaling molecules in the variation of lipid content and the modification of cell wall, and then having activated root be immunized into Journey (Figure 10).RLKs, RLPs are located in cell wall and plasma membrane, may receive root cell wall construction variation influence and it is high Expression, these signals may affect WRKY transcription factor and ERF transcription, and WRKY transcription factor is in pathogen is coerced Effect has studied very much, and the starting member for the combination pathogenesis-related proteins that course of disease correlation ERF transcription can be specific Part regulates and controls the expression of PR gene, so the expression increase of the PR gene of transcription factor regulation may be Genes For Plant Tolerance Another major reason that characteristic of disease improves.The enhancing of plant structural resistance also influences whether the variation of intracellular receptor, and then activates The anti pathologic immunity access of response, by the final expression for influencing disease-resistant gene PR gene of the regulation of transcription factor, this is resisting disease Have great importance in the research of fungal pathogens, also provides new approaches for deep excavation and research Resistant candidate genes.

We be respectively compared GbCYP86A1-1 transgenic arabidopsis disease processing with after water process three days differential gene with And the differential gene expression situation in WT between disease processing and water process, and carried out GO enrichment analysis.First from differential gene Apparent difference is shown in number, in the root of GbCYP86A1-1 transgenic arabidopsis between disease processing and water process in total There are 2541 differential genes, and wherein genes more than 1.5 times of differences only has 277, only very least a portion of gene has occurred Significant change；But have 3616 differential genes in wild type WT, and wherein gene up to 1515 more than 1.5 times of differences It is a, than being higher by 5 times or more in transgenic line.Due to the raising of disease resistance, prevent most verticillium wilt pathogen from invading or It colonizing, the root of transgenic line varies less after connecing disease, and the injury very little being subject to, internal gene tends to stable state substantially, GO is enriched to some entries relevant to growth and development；And the opposite variation of wild type is very big, main cause is that verticillium wilt pathogen is invaded Enter root and cause root cells tissue to seriously affect, while the gene of resistance needs to express to maintain normal growth and development, So be enriched to many entries, including growth and development is relevant and the relevant various entries (Figure 11 A) of resistance.Together When we have carried out the observation of free-hand section to root tissue, after choosing the root verticillium wilt pathogen processing three days of the plant of 4 weeks sizes, Longitudinal section observation is fixed, compared with other two transgenic line and wild type WT, GbCYP86A1-1 transgenosis is quasi- The root of southern mustard strain is substantially not visible the mycelial intrusion of black, this also matches (Figure 11 B) with the result of transcript profile.By Pathogen in root is difficult to invade and colonize, so the influence to root gene entirety is smaller；And wild type is due to root It infects seriously, inside is destroyed, so there is more genes to be affected into the cell, and needs to activate some resistant genes Go the further breeding of prevention pathogen.

In order to verify the accuracy of transcript, relevant qRT-PCR is carried out to transcript profile sequencing result and has been verified, water is chosen 30 genes of 1.5 times of fold differences of transcript profile sequencing result or more are verified after handling, respectively to water process and disease at The result of reason carries out statistical analysis, draws standard curve, calculates R²Value examines degree of fitting (Figure 12).

To participation cometabolism process and anti pathologic immunity in GbCYP86A1-1 transgenic arabidopsis and wildtype Arabidopsis thaliana WT The relevant gene of process has carried out the verifying of quantitative PCR, and (Figure 13) is as a result matched with transcript profile result.

Synthesis result, GbCYP86A1-1 gene can dramatically increase arabidopsis to the resistance of verticillium wilt, and the main body of resistance The intrusion for affecting root verticillium wilt pathogen now, it is disease-resistant by the physical structure and induction that influence the root cell wall of arabidopsis The activation of access assigns plant disease resistance.

(6) GbCYP86A1-1 can be expressed with the pathogenesis-related proteins of inducing cotton root

By the result of transcript profile, we have found the expression for having many PR genes positioned at downstream in transgenic line Increase, the PR gene such as reported before has very important effect (Mcfadden in the identification of signal and plant immune et al.,2001；Sels et al.,2008).In order to identify some PR genes for being related to cotton defense mechanism, in conjunction with The interpretation of result of transcript profile PR1, PR2, PR3, PR4, PR5, PR6, PR9, PR16 are under verticillium wilt pathogen induction in H7124 and army Expression pattern in cotton, these PR genes are all significantly changed by inducing expression in two cotton seeds, and induce trend It is almost the same.In order to study whether the silencing of GbCYP86A1-1 gene affects defense-related gene in root, we are further Analyze the expression of the PR gene in silencing plant.After GbCYP86A1-1 gene is by special silencing, PR1, PR5, PR16 The all extremely significant reduction of expression quantity, PR2 and PR4 significantly reduce, this may be also one of the reason of plant disease resistance weakens (figure 14).Amplimer for quantitative analysis is listed in table 6

Table 6: amplification the primer

Primer	Primer sequence (5'-3')	Purposes
			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

(7) GbCYP86A1-1 influences the cell wall structure resistance of plant and induces the activation of resistance signal's access

For GbCYP86A1-1 gene by meeting up-regulated expression after the induction of verticillium wilt pathogen, main function may be in endoplasm Net promotes the hydroxylating of fatty acid, and then activates a series of intracorporal lipid metabolism processes, polymerization and lipid by lipid The transdermal delivery of transport protein LTP increases the physical structure resistance of plant root cell wall, needs ATPase class in the process A large amount of energy is provided.While the structure changes such as the modification of the bolt matter of inducing cell wall and cell wall, can also it excite Some signaling molecules are exempted from by transmitting step by step eventually by the entirety inside the expression enhancing activated plant root of regulation PR gene Epidemic disease mechanism (Figure 15).The research is expected to using GbCYP86A1-1 be target gene by technique for gene engineerings such as transgenosis, improves The expression of GbCYP86A1-1 gene is cultivated the new germ plasm that resistance significantly improves and is applied in production.

Sequence table

<110>Agricultural University Of Nanjing

<120>the gene GbCYP86A1-1 of anti-cotton verticillium wilt and its application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1545

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

atggaattgg aaaaccttcc atttgggttc accctagcag ctgctgcaac atttgcttat 60

gcaatatggt tctatctgct agccaagagg ttaagtggtc caagggtatg gcctctggtc 120

ggtagtctac catttctttt catgaaccgg aggaggatgc atgattggat tgctagtaat 180

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 (7)

1. the GbCYP86A1-1 gene as shown in SEQ ID NO.1 or SEQ ID NO.2 is improving Cotton disease resistance and is cultivating cotton Application in flower new germ plasm.

2. application according to claim 1, it is characterised in that: using the GbCYP86A1-1 gene as target gene, pass through Gene engineering method, overexpression GbCYP86A1-1 gene cultivate the cotton new germ plasm that resistance to verticillium wilt significantly improves and in life It is applied in production.

3. a kind of method for improving verticillium wilt resistance of cotton by same, it is characterised in that: overexpression nucleotide sequence such as SEQ in cotton GbCYP86A1-1 gene shown in ID NO.1 or SEQ ID NO.2.

4. one can significantly improve the GbCYP86A1-1 gene of Cotton disease resistance, it is characterised in that: the gene is in (1)~(3) Any one:

(1) gene has the nucleotide sequence as shown in SEQ ID NO.1；

(2) gene has the nucleotide sequence as shown in SEQ ID NO.2；

(3) it is higher than 98% and nucleotides sequence functionally identical or similar with the nucleotide sequence homology of (1) or (2) described gene Column.

5. the albumen of the coding of GbCYP86A1-1 gene described in claim 4, it is characterised in that: the base as shown in SEQ ID NO.1 Because the albumen of coding has the amino acid sequence as shown in SEQ ID NO.3；The egg of the coding of the gene as shown in SEQ ID NO.2 It is white that there is the amino acid sequence as shown in SEQ ID NO.4.

6. containing nucleotide sequence GbCYP86A1-1 gene as shown in SEQ ID NO.1 or SEQ ID NO.2 or with The nucleotide sequence homology of GbCYP86A1-1 gene is higher than recombinant vector, the table of 98% and gene functionally identical or similar Up to box, transgenic cell line or recombinant bacterium.

7. recombinant vector as claimed in claim 6, expression cassette, transgenic cell line or recombinant bacterium are disease-resistant in raising cotton verticillium wilt Property and cultivate cotton new germ plasm in application.