Transformation System Of Cordyceps Cardinalis Strain C033Mediated By Agrobacterium Tumefaciens

Cordyceps cardinalis is a kind of pathogenic fungi parasitic on the larvae ofLepidoptera species. The main metabolic product is a kind of natural toxins, inbiological control of fungal insecticides can be used as important. Study on genemedicinal fungi function is an important research field in fungal genome, activeingredient and its related gene synthesis is the main research target. The mutant TIGto become the effective method for cloning of functional genes by molecular labelingtechnology, transformation of fungi by Agrobacterium tumefaciens is currently one ofthe optimal methods mutant. Therefore, this thesis Agrobacterium-mediatedtransformation system of Cordyceps cardinalis, the initial construction of the deep redworm grass T-DNA inserted mutant library, and the mutants were analyzed, resultsobtained are as follows:（1） To determine the genetic amphotericin G418and cefotaxime were C033effective screening agent and fungicide screening agent, the concentration was500μg.mL^-1. Genetic transformation system was established by using the C033strains ofAgrobacterium tumefaciens mediated by EHA105. When Agrobacterium bacteriumliquid at600nm absorption value is about0.6, Chinese caterpillar fungus sporeconcentration is105/mL, the concentration of AS is200μmol.L^-1, co-cultured withAgrobacterium tumefaciens and Cordyceps time48h, the conversion efficiency wasthe highest, which was about100transformants. And the conversion efficiency of thesame type of Cordyceps militaris30¹00transformants increased slightly.（2） The G418and NPT of II gene were screened for antibiotic and screeningmarker, subculture transformant screening again, detected by gus solution, PCRverification. That T-DNA was successfully inserted into deep red worm grass genomeDNA; the transformant strains compared with the original strain C033in colonymorphology, color, etc.. The results showed little difference, most transformant strainand original strain, great changes have taken place only a few transformant strains.Further showed that the stability of Agrobacterium mediated genetic transformationsystem of C. cardinalis strain C033.（3） The cloning and sequencing of T-DNA mutant right boundary flankingsequence of spores by using TAIL-PCR method, obtained2valid sequence, afterBLAST comparison, find one of these sequences and Aspergillus nidulans waterchannel protein gene Aqy1and GPDA gene of Saccharomyces cerevisiae similarityrespectively reached99.1%and98.2%, presumably as C.cardinalis putative genesequences. The sequence from GenBank, accession number is AEW46442.1andAB293446.1, the other2strains after repeated experiments were not obtain the idealresults, and get the right band and only1in the GenBank has homologous sequence,need further analysis. In order to study gene function, Cordyceps gene expression regulation andexpression of genetic transformation system, Genetic transformation of Cordycepssinensis is the basis of these studies, therefore the establishment of an efficienttransformation system is the key of C.militaris. At the same time, as the cloning andfunctional analysis of Cordyceps strains breeding, klgene, the technical foundationsynthesis method to illustrate the crimson Cordyceps metabolites.