关键词: 棉花, 大丽轮枝菌, 黄萎病, VdxyL3, 木糖苷酶, 寄主诱导的基因沉默

Abstract:

【Objective】 The objective of this research is to identify xylosidase genes from Verticillium dahliae and study the relationship between xylosidase genes and pathogenicity of V. dahliae, which will provide a theoretical basis for exploring the molecular mechanism of pathogenicity of V. dahliae and a scientific basis for formulating better control strategies for verticillium wilt.【Method】 All xylosidase genes were identified from the genome database of V. dahliae by bioinformatics, and their protein domain, chromosomal location and phylogenetic relationship were analyzed. Quantitative real-time PCR (qRT-PCR) was used to detect the expression pattern of xylosidase genes in V. dahliae cultured with different root exudates from resistant and susceptible cotton varieties for 0, 6, 12, 24 and 48 h. The function of one of the xylosidase genes VdxyL3 in the infection of V. dahliae was analyzed by using host-induced gene silencing (HIGS) method. The target fragment of VdxyL3 was injected into cotton, and then V. dahliae Vd991 was inoculated to those plants injected with target fragment of VdxyL3 by using root-dip approach. The phenotype of transformed plants was observed and the disease index was counted, meanwhile, the biomass of fungi and the expression level of VdxyL3 in the plants were detected by qRT-PCR technique.【Result】 Bioinformatics analysis showed that there were 13 xylosidase genes (VdxyL1-VdxyL13) in V. dahliae, whose coding sequences ranged from 1 461 to 2 544 bp, molecular weight of the encoded proteins ranged from 38.78 to 90.97 kD, and theoretical isoelectric point ranged from 4.67 to 5.89. Protein domain phylogenetic relationship analysis showed that there were 9 glycoside hydrolase 43 family members, 1 glycoside hydrolase 3 family member and 3 glycoside hydrolase 31 family members included in xylosidase genes. The chromosomal location analysis showed that the 13 genes were distributed on 6 chromosomes and no gene clusters were formed. qRT-PCR analysis showed that the expression of 6 xylosidase genes was induced by cotton root exudates. After being cultured in one or more root exudates for 6 h or 12 h, expression levels of these genes were significantly increased and then decreased. Among 6 genes, the expression level of VdxyL3 increased significantly after sensing root exudates from sea island cotton. The results based on HIGS technology showed that after 14 and 21 days of inoculation, the disease symptom of cotton plants transformed with VdxyL3 interfering fragment was more serious, and the disease index (33.3 and 83.9) was significantly higher than that of empty vector control (21.7 and 66.1). qRT-PCR analysis showed that cotton plants transformed with VdxyL3 interfering fragment had higher fungal biomass but lower expression level of VdxyL3 compared to the empty vector control.【Conclusion】 The VdxyL3 gene silencing by using HIGS technology lead to a significant decrease of cotton resistance to V. dahliae, indicating that VdxyL3 may play a certain role in the pathogenesis of V. dahliae and host-pathogen interaction.

Key words: cotton, Verticillium dahliae, verticillium wilt, VdxyL3, xylosidase, host-induced gene silencing (HIGS)