The Function Research Of Cotton Endophytic Bacteria Flagellin Against Verticillium Wilt

Verticillium wilt will cause serious damage of cotton production and lack of effective control method.Early research has shown that cotton endophytic bacterial Pseudomonas strains 841 P-3 can cause cotton defense signal and defense production,stimulate the cotton defense responses,improve the resistance to verticillium wilt,but the mechanism is not clear.Plants can produce inducible resistance by recognizing different types PAMP of bacteria,the flagellin Flg22 short peptide is one of the most deepest study currently.In order to study whether 841 P-3 strains in the presence of flagellin gene,and study the relationship between these genes and 841 P-3 in the induction of cotton verticillium wilt.Four flagellin genes,were isolated from the Pseudomonas strains 841 P-3 in the genomic DNA.One of these genes,FlgL,was shift to E.coli BL21(DE3)expression through prokaryotic expression system and reorganization the purified flagellin FlgL.FlgL could activate the hypersensitive response(HR)and systemic acquired resistance(SAR)in tobacco plants.In order to investigate the roles and mechanisms of FlgL in cotton resistance against V.dahlia,we tested the disease-resistant cotton defense related material and defense enzyme activity,detected the cotton PR genes and the key gene level of SA,JA signaling pathways using RT-PCR technique.In this paper,the experimental results are as follows:The four flagellin genes,FliC、FlgL、FliS、FlgK was clonined from Pseudomonas aeruginos 841 P-3 genomic DNA,and purified through the prokaryotic expression system,the molecular mass of the proein is 46.9 kD,pI=4.34.Biological activity showed that FlgL can lead to tobacco leaf produce hypersensitive response(HR).FlgL flagellin triggered different resistant varieties of cotton defense responses and activated early signaling events:reactive oxygen species;secondary metabolite generation such as callose generation and deposition.The activity of phenylalanine ammonia enzyme(PAL),peroxidase(POD)and polyphenol oxidase(PPO)increased in different degrees,and these enzyme activities in resistant varieties increase later than susceptible cultivars.The maximum activity of PAL appeared at 4 to 6 d and was 2 times of control,but there was not significant between them;The maximum activity of POD appeared at 6-8 d,the highest activity in resistant varieties was 3.5 times of the susceptible cultivars;PPO activity was significantly higher.The early reaction shows that FlgL is a functional protein elicitor.FlgL induced systemic acquired resistance(SAR)in tobacco against Verticillium wilt.FlgL could obviously improve the resistance to verticillium wilt in cotton,and the effect of resistant varieties cultivars was stronger than susceptible cultivars,the relative control efffects were 43.42%and 31.59%respectively at 30 days post FlgL treatment.RT-PCR revealed that resistance-related genes PR-1 and PR-10 were upregulated expression after FlgL treatment in cotton,the results show that the induced FlgL cotton defense genes PR-1 and PR-10 raising expression,FlgL could induce cotton resistant cultivars and susceptible cultivars produce system disease resistance.Detection of the higher expression of key genes in salicylic acid signaling pathway marker genes NPR1,POD4,no obvious change of the key gene of jasmonic acid marker genes AOS and the key gene of ethylene signaling pathway marker genes ERF5,but the key LOX gene expression of jasmonic acid way is raised,demonstrated that SA signaling pathway is a main pathway involving in the defense response induced by FlgL.FlgL gene was inserted into the transformation vector PB1121 vector,the recombinant PB1121-FlgL Arabidopsis inflorescence via Agrobacterium tumefaciens EHA105,the T0 generation transgenic Arabidopsis plants were obtained by sereening on MS solid medium.This study cloned four flagellin genes from bacteria,purified recombinant flagellin by prokaryotic expression flagellin gene for the first time,and verified that the flagellin can also increase the cotton defense reaction and enhance the resistance to verticillium wilt.The research not only a new way for controlling cotton Verticillium wilt,but also provide the theoretical basis for clarifing resistance mechanism of the cotton endophytic bacteria 841 P-3 induced resistance.