Study On The Pathogenic Factors And Functions Of Anthrax In Walnut Based On Transcriptome

Walnut(Juglans regia L.)is an important economic forest species in China.However,the walnut anthrax caused by Colletotrichum gloeosporioides severely reduced yield of walnut and caused great economic loss.Therefore,it is important to clarify the molecular mechanism of walnut anthrax.In this study,the transcriptome sequencing technology based on Illumina sequencing platform was used to carry out transcriptome sequencing on the mycelia of Colletotrichum gloeosporioides strains HTTJB-6 and HTTJB-28 with different pathogenicity.Transcriptome data of two samples were analyzed by protein-coding gene expression level analysis,differentially expressed gene analysis,variable shear analysis and SNP/INDEL analysis,and disease-related genes were screened by combining with bioinformatics.The effector protein genes,which may be pathogenic genes,were selected to verify their functions by knockout.The results are as follows:1.Transcriptome libraries of HTTJB-6 and HTTJB-28 mycelia were prepared,and14.94 g of Clean Data was obtained by sequencing.The analysis results were as follows:Through the analysis of differentially expressed genes,559 differentially expressed genes were obtained.All the differentially expressed genes screened were annotated with GO function,and we found that 272 differentially expressed genes were annotated into40 secondary GO functional entries.Catalytic activity was the function item with the most enriched differentially expressed genes,with 189 differentially expressed genes enriched.Through the enrichment analysis of metabolic pathways,we find 14 differentially expressed genes are involved.Genes related to lipid metabolism pathway and Xenobiotics biodegradation and metabolism pathway may be related to the pathogenicity of colloidal anthrax.Ten of the 59 different genes assigned to the KEGG pathway were selected and their expression trends were verified by q RT-PCR,and the detection results were consistent with the expression trends of transcriptome sequencing.2.Using bioinformatics methods and transcriptome sequencing results,five effector protein genes(CFEM12538,CFEM15397,CFEM11946,CFEM11363,CFEM05962)were screened as target genes,and gene knockout vectors were successfully constructed.After gene knockout by agrobacterium tumefaciens transformation,it was found that there were too many single colonies to pick up.Moreover,the protoplast preparation method was used for transformation,and multiple single colonies were obtained.PCR detection showed that some of the single colonies had hypopycin resistance gene fragments inserted,but the target gene was still present,and the result was ectopic inserted single colony.