Establishment Of Genetic Transformation System Of Cordyceps Militaris And Creation Of Auxotrophic Strain

CRISPR is a new type of gene editing technology.It is compared to the precise molecular scalpel,and has great potential and prospects for the human disease gene mining,disease targeted therapy,agricultural crop varieties,and directional breeding.This technology is simple and easy to operate,and the experimental cycle is short,cost savings,easy to carry out in the laboratory.CRISPR technology has been used in plants,animals and human cells.Cordyceps militaris as a food and medicine fungi,many effects are close to Cordyceps sinensis,with slow fatigue,soften the cardiovascular,anti-tumor,radiation and other effects,the industrialization of Cordyceps militaris fruiting body system has been established.The genetic transformation system was established in Cordyceps militaris for the first time in this experiment,and the gene of URA3,a key enzyme gene for uracil synthesis in Cordyceps militaris,was first invented by CRISPR technique to make it a kind of genetic modification system.New screening markers,in order to further study the metabolism of Cordyceps militaris,bacteria optimization laid the basis for genetic engineering.In this study,the orthogonal transformation method was used to optimize the transformation conditions of Agrobacterium tumefaciens-mediated Cordyceps militaris,and the Agrobacterium tumefaciens mediated protoplast transformation system was established for the first time.The GPD promoter was successfully cloned and successfully constructed on the knockout vector pFGC-pcoCas9,which thus realizes the transformation of the plant knockout vector and finally obtains the knockout vector suitable for Cordyceps militaris.By using the Agrobacterium tumefaciens genetic transformation method,the knockout vector was transferred into protoplasts of Cordyceps militaris,and finally the mutant was obtained,and the genetic and genetic transformation were achieved.The specific results of the experiment were as follows:1.The protozoan of Cordyceps militaris was cultured for 4 days by the combination of snail enzyme and lysozyme,and the ratio of 1: 1,Extraction averaged 1×107 cells / mL.2.Through the Antibiotics experiments on Cordyceps militaris protoplast sensitivity,the optimum screening concentration combinations were obtained:hygromycin + kanamycin: 650 mg / L + 300 mg / L.3.The effects of genetic transformation of Agrobacterium tumefaciens on transformation efficiency were studied by orthogonal method and verified by fluorescent protein gene.The optimum conditions were as follows: the concentration of acetosyringone was 200 ?M / L,the volume ratio of Agrobacterium and Cordyceps militaris protoplast was 100:1,and the co-culture time was 2 days.4.The GPD promoter was cloned and successfully constructed on the knockout vector pFGC-pcoCas9,and the gRNA of the URA3 gene sequence was ligated into the vector to obtain the vector suitable for the experiment.5.By Agrobacterium-mediated transformation of Cordyceps militaris,the knockout vector was successfully transferred into Cordyceps militaris and obtained transformants.6.The positive transformants were washed with 0.9 % saline to perform subculture four times.The results showed that the transformants were stable and still susceptible to antibiotics.It was proved that Agrobacterium tumefaciens was successfully established by genetic transformation of protoplast of Cordyceps militaris.