Prokaryotic Expression Of Pectate Lyase Gene And Pathogenic Detection Of Pythium Ultimum

Pythium ultimum is one of the common pathogenic Pythiums which can infect somekinds of plants and lead into pathological changes. It has a broad host spectrum,refers to,Leguminosae Asteraceae, Cucurbitaceae, Solanaceae, Rosaceae and so on, the hostsalways show some rotten symptoms in the late once infected by Pythium ultimum. Atpresent, vegetable growing areas in Shandong, the Pythium ultimum-induced disease ismore and more serious, especially on tomato (Solanum lycopersicum). Tomato can beinfected throughout the growing period, and it is the most serious disease at the seedlingstage infected by Pythium ultimum. Furthermore, with the development of greenhousevegetables, it occurs much more frequence as the greenhouse provides more suitableconditions for the growth of the disease.In order to reveal the pathogenesis of Pythium ultimum to guide the production practiceto prevention and treatment of disease caused by Pythium ultimum. In this study, weanticipated to analysis the physical and chemical changes during the pathogenic processsince it caused infection from Pythium ultimum. It was separated and identified which wasthe pathogen of tomato basal stem rot, as well as analysised of pectate lyase which mayplay an important role during the pathogenic process of Pythium ultimum on tomato.Thenthe sequence of pectate lyase gene was cloned from Pythium ultimum, to carry a vitroexpression based on a prokaryotic expression vector, futhermore, in this research, aknockout vector was constructed successfully to pectate lyase gene, which laid thefoundation for further study of the function of pectate lyase gene. It was also put forwardsome recommendations on prevention and treatment for Pythium ultimum-induced diseasesin vegetable growing areas of Shandong.Results of the study are as follows: 1. Separeted the disease-causing pathogens of basal stem rotten from the infectedtomato plants, and identified the pathogen by morphology——inducing the pathogen withPetri`s solution then observed the special structuls which referred to hyphae swollen bodyect. The ITS sequence of the pathogen was cloned with the primer pair——ITS1/ITS4,which is proved to be Pythium ultimum.2. The Pythium ultimum was induced by pectin, resulting in a high activity of pectatelyase, combined with fluorescence quantitative analysis of pectate lyase from Pythiumultimum on tomato during the pathogenic process;3. A sequence of pectate lyase-with a length of726bp was cloned with Pythiumultimum genome DNA, which was named Pel. Then a prokaryotic expression vector namedpEASY-Pel was contructed, carrying the Pel sequence, and was thransformed into E.coliBL21, with a final concentration of1mM IPTG induced, expressed a size of about26kDtarget protein, indicating that the protein was pectate lyase;4. From the Pythium ultimum genome, the pectate lyase gene left arm and right armsequences of each1000bp were cloned, to build pBI-G3C, the pectate lyase knockoutvector; used for a further deletion mutations of pectate lyase gene.