Study On The Functions Of Core Regulatory Genes In The Spore-producing Pathway Of Purple Spor

The filamentous fungus Purpureocillium lavendulum is an important biocontrol agent for plant-parasitic nematodes.To investigate the regulation genes involved in the sporulation of P.lavendulum.The PlAbaA,PlWetA,PlFadA and PlPkaA genes was knocked out and complemented using the homologous recombination techniques.Subsequently,we investigated the specific function of these regulatory gene by examingthe number of conidia,the asexual conidiogenous structure and the capacity of spore germination.To explicit the biological functions of these genes,further phenotypic examination was performed on the variation phenotypes of the above strains.This work established the foundation for further elucidating the development mode of conidiogenesis in P.lavendulum.The main results of this stuty were described as follows:1 The sporogenesis of P.lavendulum was regulated by PlAbaA,PlFadA and PlPkaA geneThe morphology of colonies,the conidium structures,the ability of sporulation,the germination rates of spore and stress resistance among obtained mutants and PlKu80 strain were compared.The results showed that the PlFadA and PlPkaA mutants produced more conidia than PlKu80 strain.While both PlPkaA and PlFadA mutants showed no difference with PlKu80 on the conidiogenous structure.In contrast,the PlAbaA knockout strain did not produce conidia,the conidial structure was incomplete,with a defect in development of the phialide.The colony of the mutant was white and was more compact than its parent strain.The PlWetA null mutant forms offwhite conidia and shows an intact sporulation structure through SEM.When investigated with TEM,the PlWetA knockout strain showed some different structures,the germination rate decreased,and showed higher sensitivity to external stress compared to the ?PlKu80 strain.2 Trehalose production in P.lavendulum was influenced by the PlWetA geneThe content of trehalose in mycelia and spores was determined.In the mycelia,the concentration of trehalose displayed almost no difference between ?PlWetA and ?PlKu80.In the conidia,the concentration of trehalose in PlWetA knockout strain were much lower than in the ?PlKu80 strain and the PlWetA complement strain.Northernblot analysis of trehalose synthesis related genes revealed that in mycelia,mRNA levels of tps1,tps2 and tps3 were not affected by knock out of Pl Wet A,whereas in the conida,the expression of the three genes decreased in ?PlWetA strain compared to the ?PlKu80 strain.3 The deletion of PlWetA,PlBrlA,PlAbaA genes influence the production of the secondary metabolite leucinostatin ABWe anaylized the secondary metabolic products in the strains ?PlBrlA,?PlAbaA,?PlWetA and the reference strain PlKu80 knockout strain by HPLC.We found that the metabolite profile of PlWetA and ku80 knockout strain had a specific peak of leucinostatins AB,compaired with the internal standard.However,no corresponding peak in both the mutants ?PlBrlA and ?PlAbaA,and almost all the metabolite peaks have been reduced or decreased.The main innovation of this paper:The regulatory genes involved in sporulation of P.lavendulum were studied for the first time,which may make a base for further studies of biocontrol against nematodes.