The Cloning And Initial Function Analysis Of StUBCc Gene In Solanum Torvum

Verticillium wilt is one of the main soil-borne diseases of eggplant.It can reduce the production of eggplant resulting in serious economic losses. Cultivars are lack of resistant materials to verticillium wilt. High resistant materials are almost wild species,especially Solanum torvum. Cloning anti-verticillium wilt genes from Solanum torvum has significance to resistance breeding.This research is based on Solanum torvum suppression subtractive hybridization(SSH) library induced by verticillium wilt fungus.159 ESTs sequences from SSH were aligned again to vertify the differential expression. After it, RACE was used for the cloning of verticillium wilt resistance-related gene and then VIGS was utilized to study the function of the cloned gene.The main results were listed as follows:1. Utilizing qRT-PCR analyzed the 12 ESTs related to resistance from SSH library to vertify the differential expression.The result showed that the expression of verticillium wilt fungus infection was different with the control.During 2 hours post infection,the expression of each EST had little difference compared with the control.After 4 hours post infection, the expression was higher and belonged to upregulated expression.2. The full-length cDNA(756bp) was obtained from Solanum torvum using rapid amplification of cDNA ends(RACE) technology.This gene had a 459 bp opening reading frame with 99% identity to ubiquitin-conjugating enzyme E2-2(UBC2)protein in Vitis vinifera,named StUBCc. The gene was close to grape in the evolutionary tree. Result of qRT-PCR showed that the expression of StUBCc gene treated by verticillium wilt fungus was higher than the control treatment,and the expression of the newly isolated StUBCc gene was up to the highest level at 6 hours post infection,which was 6.79 times more than the comparison. It indicated that the gene may participate in the process of early reply to pathogen infection.3. Utilizing VIGS verified the function of StUBCc gene.301 bp cDNA was selected to connect in an vector to construct StUBCc gene silencing expression vector. And then,expression vector was transfected in Solanum torvum seedlings by Agrobacterium-mediated method.The result showed that the 66% plants grew weaker than the control and the leaves turned wrinkled.The expression of StUBCc gene fell down. At 4 weeks post infection, the expression,which was 37.43% of the control,was down to the lowest. The leaves of nearly 33% treated plants turned yellow post infection by verticillium wilt fungus.