Virulent Analysis Of Adenylate Kinase And Oligosaccharyl Transferase STT3 Subunit In Verticillium Dahliae

Verticillium Wilt of cotton, named "cotton cancer", is a kind of soil-borne, systematic vascular disease, which produces the heavy influence of cotton production and cotton fiber qualify. The pathogenic fungus was Verticillium dahliae. The V. dahliae was easy variation to generate new physiological strains, which brought enormous difficulties and nondeterminacy. The research of V. dahliae has being a hot topic for a long time. The host-induced gene silencing (HIGS) technology was previously screened the developmental and virulent genes of highly virulent strain V991. A series of tobacco rattle virus (TRV) RNA interference (RNAi) vectors were constructed. After the interaction between Nicotina benthamiana and fungi and statistics of disease index,20 genes was prediceted to display the certain relationship with virulence. In this study, adenylate kinase (AK) and oligosaccharyl transferase STT3 subunit (OST STT3) were selected to furtherly investigate the genes' function and boarden the resistant genes data in plant. The main results of this research were listed as follows:1. Firstly, larger number of experimental seedlings was sreened using the HIGS system. 10 days post Argo-infiltration of AK and STT3 individual, the seedlings were inculated by root dipping in 106 spores/mL solution. Then, the disease index was recorded. The fungal biomass and expression level of targeted genes were determined by qRT-PCR.2. By the melted PCR, the upstream and downstream of targeted gene were fused with Hygromycin resistant expression cassette. The fragment was cloned into pGKO2 vector as deletion plasmid via BP reaction. The expression cassette of neomycin resistance and targeted gene was inserted into pCAMBIA1302 as complementary plasmid. Menawhile, the open reading frame (ORF) of AK was replaced by GFP ORF. In the end, the transformants were obtained by Agrobacterium tumefaciens mediated transformation (ATMT).3. The seedlings of N. benthamiana were inoculated by the spores of wild type (WT), ak mutant and complementary strains. The disease index was recorded from 10 days post inoculation (dpi) to 12 dpi. The roots of seedlings inoculated by Vd-GFP or mutant GFP strains were observed to analysis the difference after the deletion of targeted genes.4.The spores of wild type (WT), ak mutant and complementary strains were dropped on the Czapek-Dox medium. The plates were treated by different adverse situation. Meanwhile, the spores of wild type (WT), stt3 mutant and complementary were dropped on the Czapek-Dox media with different carbon. After two weeks, the fungal characterics were detected, including colony diameter and spore number.5. The stable inherited plasmids produced dsRNA were constructed to transform the N. benthamiana. The resistant levels were assessed by disease index and molecular detection.Collectively, the AK and STT3 biological functions were furtherly analyzed by the transformants of mutant and complementary and positive plants. The research was focused on the key genes involved in the fungal development and virulence, which would contribute to provide experimental dada to prevent the agricultural pest and disease.