| Cotton is one of the important economic crop in China, but its yield and fiber quality is seriously affected by the Verticillium wilt. Verticillium wilt in China is mainly caused by Verticillium dahliae, the soil borne disease, so researches of the mechanism of cotton verticillium wilt become the key of the cotton disease-resistance breeding. In this study, we screened all the WRKY genes of this family and toke bioinformatics analysis on the basis of cotton genome sequencing of Dt. After analyzing the transcriptome database information of Verticillium wilt of cotton and Real-time PCR results, we screened and cloneed WRKY46,a disease resistance related gene. Via bioinformatics analysis of the WRKY46, analyzing differential expression levels of cotton infected by Verticillium dahliae or induced by phytohormone, and observing the phenotype after virus induced gene silence, we obtained the following results:1. In this study, we identified 110 WRKY genes in cotton D genome, including 101 regular and 9 non-regular type genes. All of these genes were characterized through analyses of gene duplication, chromosomal location, structural diversity, conserved protein senquences and phylogenetic relations. The results showed that 12 times of gene duplication event occurred in WRKY gene family involving 29 genes. And there were 3 gene clusters were found in WRKY genes. WRKY family members can be divided into three major groups, and their conserved domain are polymorphic.2. Among 21 candidate WRKY genes, expression analysis suggested that the expression levels of 14 WRKY genes were up-regulated and the expression levels of 1 gene(WRKY103) were down-regulated after Verticillium dahlia inoculation. Besides, there were 6 WRKY genes with no obvious expression changes. Expression analysis illustrated that WRKY38, WRKY46 and WRKY104 expressions were up-regulated, while WRKY40, WRKY63, WRKY72 and WRKY76 expressions were down-regulated after salicylic acid treatment. According to the expression data of the transcriptome database of Pima90-53(a high resistance variety), Nongda 601(a disease-resistant variety) and CCRI8(a susceptible variety), we found that the expression of WRKY46 is significantly higher in sea island cotton than that of upland cotton.3. The ORF of WRKY46 were cloned from Pima90-53 and Nongda 601, respectively. There is only one amino acid mismatch of the proteins sequences, and this site is not in the conserved domain of WRKY46. Genome sequence of WRKY46 contains two introns. WRKY46 is a soluble protein which has a homology of Gossypium barbadense and Jatropha curcas. It was located in the nucleus without transmembrane structural domain and it does not contain a signal peptide.4. Induced by Verticillium wilt, the expressions of WRKY46 were significantly upregulated in the three varieties of cotton. WRKY46 expression was up-regulated in Pima90-53 when treated with SA, Me JA or ET, respectively, indicating that the expression of WRKY46 was affected by these three hormones.5. After silencing the WRKY46 gene in Pima90-53 and Nongda601 by virus induced gene silencing, the plants in which WRKY46 expression was obviously reduced appeared symptoms earlier. When the disease break out, the disease index of the silencing plant was 43.7 while the control was 35.3 in Pima90-53. These results showed that the silencing plants had a declined resistance with the control.6. After silencing the WRKY46 gene in Pima90-53 by virus induced gene silencing, the expression of marker genes of phytohormone signaling pathways were detected after Verticillium dahliae inoculation, respectively. AOS expression was up-regulated obviously, while EDS1, PR1, PR5, PR4 and ACS expressions were significantly down-regulated. These results showed that WRKY46 was involved in SA, JA and ET signalling network. |
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