Expression Analysis Of Cotton Anti-Verticillium Wilt Related Genes And Preliminary Functional Research Of GhPR10

Verticillium wilt of cotton is a major disease which causes reduction in both the quality and quantity of cotton and results in substantial economic losses. Applied with combination of biotechnology and traditional breeding, we can screen cotton resistance-related genes, cultivate new breed directionally which is the easiest way to control disease.We use RT-PCR to test 8 pathogen toxin, salicylic acid and absisic acid infected cotton seedlings, analysis 22 cDNA sequences which acquired by suppression subtractive hybridization, homolog cloning and so on. 19 out of these 22 genes can be induced by Verticillium dahliae, 17 can be induced by Aspergillus niger van Tiegh and GhPR10, GhLRR, GhPRG, GhGT, GhCOX, GhVDRG11 can response to infection of most of pathogen, VerticiUium wilt of cotton resistance-related gene GhVE1 can be induced by V. dahliae specifically. GhPR10, GhGGH and GhVDRG11 are transient expression genes which can be detected in 10 minutes, 14 genes can be induced during 30min to 90min. 15 genes are co-expressed after 6 hours, comprising GhVE2, GhEH and Verticillium wilt of cotton resistance-related gene GhVE1. In addition, GhEH, GhZFP, GhVE1, GhVE2, GhLRR and GhGGH expressed differently in Gossypium hirsutum L. var. Junmian 1, Gossypium hirsutum L. var. Zhongmian 12, Gossypium barbadense L. cv. Hai7124 and Gossypium hirsutum L. var. L12 which have different resistance to Verticillium wilt of cotton. Hormone induction result indicate that GhPR10 can be induced by SA and ABA, GhDIR and GhGT which involved in transglycosylation can be induced by SA, ABA up regulated GhVEland down-regulation the expression of GhTMR. Combined the result of the expression, we predict that there are two stages of the resistance of cotton to Verticillium wilt: transient basal resistance and lagging specific resistance. Genes involved in Basal resistance expressed in 30 minutes and lagging specific resistance genes expressed at 6 hours. GhPR10, GhPRG, GhCPH, GhPDR and GhTMR are core genes in transient basal resistance pathway while GhVE1, GhVE2 and GhLRR are core genes in lagging specific resistance pathway. ABA and SA regulate core genes of basal resistance and lagging specific resistance genes.RACE and TAIL-PCR were employed to screen candidate gene GhPR10, GhEH, GhPRG, GhZFP, GhERF and GhDIR. GhPR10(DQ123838) contained one intron spanning 108 bp, cDNA is 774bp in which a 474bp ORF encoding a 157aa protein, 1537bp upstream of ATG could be a predicted pathogenesis-related protein Bet vâ… family gene. GhEH(DQ123837) cDNA is 1571bp, containing a 1160bp open reading frame, encodes a 315aa protein molecule which predicted to be a epoxide hydrolase gene. Four introns locate in it, 119bp, 81bp, 83bp and127bp respectively. The GhERF(DQ372577) cDNA comprised 1319 bp and included an ORF of 516bp, encoding a 171 aa protein which predicted to be a ethylene response element binding protein. GhZFP(DQ372575) cDNA is 768bp composing an ORF long 516bp, coding for 171 amino acids which could be a zinc finger proteins. GhPRG (DQ372577) cDNA sequence is 1020bp coding a 264 aa protein which may be component of cell wall. GhDIR cDNA is 765bp coding a 176aa protein which may involved in lignify.Agrobacterium-Mediated Transformation was used to confirm the function of GhPR10 on tobacco and Arabidopsis and 34 GhPR10 transgenic tobaccos were got. Identification the resistance using detached leaves indicate that the overexpression of GhPR10 can increase the resistance of tobacco to Verticillium wilt of cotton. The total amount of flavone in the transgenic tobacco leaves doubled compared with the control in one gram, the highest is 1.10mg and 0.883 average, but the control is only 0.375mg, indicate that GhPR10 can influence the level of plant antimicrobial compound, such as flavonid. The transgenic Arabidopsis also indicated that GhPR10 can enhance the tolerance of V. dahliae invasion and salt stress. In addition, GhPR10 can influence plant development by changing the phenotype of tobacco and Arabidopsis, such as grows slower, while the leaves of transgenic Arabidopsis is round, much more lateral branches and have reduced apical dominance.