Identification Of Verticillium Wilt Resistance ESTs In Solatium Torvum Using Suppression Subtraction Hybridization

Eggplant is one of the most important vegetable in china,and has an important position in Chinesevegetable production.The eggplant production and areas rank first place in the world.With limitedvegetable production areas, the soil-borne diseases in eggplant production has become more and moreserious due to the multiple cropping index increased. Verticillium wilt is common and difficult tocontrol. The disease decrease the eggplant quality and quantity,so it is important to understand themolecular mechanism and to obtain related resistant gene and analyse the gene’s function. Theexperiment includes to construct SSH libraries of resistance to verticillium wilt from Solanum torvum，and to obtain related gene to resistant to Verticillium Wilt in eggplant, and to study the molecularmechanism. The main results were as follows:(1) One forward and one reverse cDNA library were constructed by suppression subtractivehybridization （SSH）. Wild relative species eggplant Solanum torvum is used as material in thisexperiment, the material under Verticillium dahliae treatment as the experimental group, the materialunder sterile water treatment as control. there were199different expression unique ESTs in the forwardand reverse libraries after removing and screening the2376clones in the libraries. Blastx analysisshowed that there were103of these unique ESTs were homologous to known function protein in databank, and55of these unique ESTs had matched sequence with unknown function,and41uniqueESTs did not find matched sequence.GO functional annotation showed that the total of199uniqueESTs divided into three categories and24subcategories.the result showed that S. torvum withVerticillium dahliae infection induced resistance differentially expressed genes involved in multiplephysiological pathways, the expression of the resistance genes may be regulate by some combination ofprotein and transcription factor.(2) Based on the BLAST results of differentially expressed Unique ESTs from suppressionsubtractive hybridization cDNA libraries,there were8differentially expressed Unique ESTs wereselected which maybe relate to Verticillium Wilt Resistance,and the real-time PCR were used toadvanced research them,the Real-time PCR results showed that4Unique ESTs （Z471、Z992、Z778、F25）were up-regulated genes, a unique ESTs （F1155） was down-regulated gene, the rest of3uniqueESTs （Z32, Z882and F1245） under different treatment conditions did not have regularity.(3) According to the results of the real-time PCR,an Unique ESTs （Z471） which is up regulatedexpression was cloned from Verticillium dahliae and sterile water treatment Solanum torvum SeedlingRoots utilizing RACE technology.The full length cDNA sequence were the same under differenttreatment conditions.This gene, named StPTI5.StPTI5gene has a522bp open reading frame, coding173amino acids，the5’end has a43bp non-coding region, the3’ end has171bp non-coding regionincluding the PloyA tail. Its molecular weight is19.4349KD and theoretical isoelectric point PI was 7.85. It contained an AP2domain which had90%homologus with disease related activatingtranscription factor of tomato.