| Cotton Verticillium wilt is one of the primary diseases that affects the production and quality of cotton, so it is important for cotton anti-Verticillium breeding to screen the resistant related genes and elucidate the mechanism of anti-Verticillium of cotton. In this research, the full-length cDNA sequence of Vdrg2 from SSH was cloned. The results of bioinformatics analysis indicated that this gene shares high homologous with protein phosphatase 2C (PP2C) that participate in ABA signal transduction and was designated as GhPP2C. The timing expression characteristics of GhPP2C were examined in different cotton varieties treated with crude extraction toxin from different virulence Verticillium dahliae strains. The Arabidopsis thaliana transgenic plants of GhPP2C were obtained by the method of Agrobacterium-mediaed transformation.The 1471bp full-length cDNA sequence of GhPP2C gene was cloned through 5'RACE. The open reading frame(ORF) was 1251bp in length and deduced protein sequence was 416 amino acids .The analysis of DNA sequence indicated that GhPP2C had 3 introns, i.e. 98bp,341bp and 96bp. The results of BLAST showed that GhPP2C belonged to the PP2C family and shared 59% similarity with ABA-Hypersensitive Germination 3 (AHG3).The seven-day aged seedlings of three different resistant cotton varieties were used to test the expression characteristics of GhPP2C after treating with different virulence crude extraction toxin from Verticillium dahliae strains VDK 114 and VDK991 at 4 time points (2h,6h,12h,24h) by RT-PCR.The results indicated that when treated with high virulence strain VDK991, GhPP2C in resistant variety Island cotton 06 expressed from 12h to 24h, whereas in tolerance variety zhongmian 12 and susceptible variety zhongzhi 86-1 GhPP2C only expressed at 2h. When treated with high virulence strain VDK114, the expression of GhPP2C showed more complicated. GhPP2C was expressed at 6h to 24h in resistant variety and 2h to 12h in tolerance variety and only 12h in susceptible variety. Semi RT-PCR results showed that there was no significant relationship between the GhPP2C expression level and the variety resistances. In summary, there was negative relative between the timing expression characteristics of GhPP2C and the resistances of cotton variety, i.e. the expression time of GhPP2C was lagged in resistant variety when treated with VDK991 and was supperessed longer time in susceptible variety. The expression analysis of GhPP2C in zhongmian 12 treated with ABA, ET and SA indicated that, GhPP2C was suppressed in seedling stem by ET, but not in leaf. The normal expression of GhPP2C was not affected by ABA and SA.The predicted GhPP2C active site was substituted (Aspl52 to Glyl52) by the method of site special mutation PCR, and the mutanted gene was named as GhM152. The expression vector of GhPP2C and GhM152 were constructed, the recombinant plasmid were designated as pBIPP2C and pBIM152 respectively, and transformed into Arabidopsis Columbia ecotype. The transgenic plant with pBIPP2C and wild type seeds were planted on 1/2 MS solid medium supplemented with ABA or GA for testing germination and growing. The results dedicated that both transgenic and wild type plants could germinated but could not grow on the medium supplemented with ABA in the concentration of 20～100μmol/L. When treated with low ABA concentration of 1～2.5μmol/L, transgenic plant could germinate and grow normally but the growing of wild type was suppressed. These phenotypes displayed GhPP2C could elevate the tolerance of transgenic plant to ABA. In addition, there was no significant difference in germination and growing between transgenic plant and wild type on the medium with GA (20～100μmol/L).
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