Cloning Of Candidate Effector Protein Genes And Function Preliminary Verification Of Two Genes

Valsa mali the pathogen causing destructive apple tree canker in eastern Asia,such a kind of disease was first reported in Japan in 1903.it leads to terrible lose in products and little is known about its essence.Recent research shows that effector proteins play a very important role during Valsa mali interacting with its host.To research about those possible effector proteins,the first step is to clone those genes from genome,in this study we predict 119 candidate effector proteins from genome and try to construct the 119 candidate effector proteins gene’s pool which could support the later research,what is more,we chose the homologous gene of Nis1 and Neps in the pool for the preliminary verification of its function.1.TA cloning and one step cloning are chosen to clone 119 genes to PGR106 and PCHF3 vector respectively.Finally,78 genes are cloned to PGR106 vector and 78 genes are cloned to PCHF3 vector.The candidate effector proteins gene’s pool of valsa mali is preliminary constructed which could be used for the later research.2.Homologous gene of Neps(VM1G＿10052)and homologous Nis1(VM1G＿07228)are chosen from the pool for the later research and preliminarily verify their function,transient expression mediated by agrobacterium tumefaciens shows that VM1G＿07228 could induce obvious necrosis on the Nicotiana benthamiana,while VM1G＿10052 could not.3.VM1G＿07228 is chosen for the later gene knockout with PEG mediated protoplasts transformation,2 positive transformants(#45 and #106)are selected from 178 colonies.Following pathogenicity test on both twigs and leaves shows VM1G＿07228 do not contribute to virulence of valsa mali.