Virus-induced Gene Silencing-based Analysis Of Genes In Response To Verticillium Dahliae And Function Characterization Of GhB2 In Gossypium Hirsutum L.

Cotton is one of the most important crops in the world, because it not only provides fibre for the textile industry, but also plays a role in oil industries with oil-enriched seeds. Verticillium wilt is one of the most serious threats to cotton growth, causing significant decrease in yield. Verticillium wilt is caused by the soil-borne fungi, Verticillium dahliae(V. dahliae), which are widely distributed in agricultural soils with a broad host plants. The dormant microsclerotia may remain viable in the soil for more than 10 years. Therefore, breeding and cultivation of resistant varieties become the most economical and environmental friendly way to control the disease. However, Gossypium hirsutum(G. hirsutum) varieties with resistance to Verticillium wilt generated by traditional breeding method is still very limited.The 23 candidate genes were selected based on high-throughput RNA-seq analysis of Zhongzhimian KV-3 upon V. dahliae infection. 20 candidate genes are up-regulated genes upon V. dahliae infection; 3 candidate genes are down-regulated genes upon V. dahliae infection. We investigate the 23 candidate genes in response to V. dahliae infection, and the results are as follows:1. Real-time quantitative PCR was performed to check the expression difference of 23 candidate genes in susceptible variety 86-1 and resistant variety KV-3. The results showed that the expression level of 20 up-regulated genes were induced in both susceptible and resistant variety, and expression level of up-regulated genes increased faster and sooner in resistant variety Zhongzhimian KV-3 than in susceptible variety 86-1 upon V. dahliae infection. The expression level of 3 down-regulated genes were suppressed in both susceptible and resistant variety, and expression level of down-regulated genes decreased more greatly in resistant variety Zhongzhimian KV-3 than in susceptible variety 86-1 upon V. dahliae infection.2. 23 candidate genes were investigated via virus-induced gene silencing(VIGS) using cotton leaf crumple virus(CLCrV) vector. The results found that silencing 20 up-regulated genes significantly enhanced resistant variety susceptibility to V. dahliae and silencing 3 down-regulated genes significantly increased susceptible variety resistance to V. dahliae.3. The full-length of GhB2 were cloned using rapid-amplification of cDNA ends(RACE), including 969 bp Open Reading Frame(ORF) and encoding 322 amino acids. The protein belongs to DCD superfamily due to that it contains conserved a FGLP and a LFL motif at the N-terminus and a PAQV and a PLxE motif towards the C-terminus of the domain. It shares high similarity with Gossypium raimondii, Gossypium arboreum and Theobroma cacao. The molecular weight is 35.98 kD, and theoretical pI is 8.75. It also doesn’t possess signal peptide and transmembrane structure. The GhB2 is expressed in root, stem, leaf and flower, and accumulation of the GhB2 in flower was lower than in other organs.4. The overexpression vector pPZP111-eGFP-GhB2 was transferred into wild type Arabidopsis thaliana via floral dip method. The average DI of pPZP111-eGFP-GhB2 overexpression plants was 16.95, while the average DI for control(pPZP111-eGFP) and wild type plants were 40.04 and 34.56, respectively. The results showed that overexpression plants enhanced resistance to V. dahliae and plants showed a lighter symptom than the control plants, indicating that GhB2 plays an important role in response to V. dahliae infection.