Optimization Of The Transformation System And De Novo Characterization Of Transcriptome Of Aschersonia Placenta

Aschersonia placenta is an environmentally friendly entomopathogenic fungus with an enormous potential.However,there have been no studies on optimization of the transformation system and de novo characterization of transcriptome of A.placenta at home and abroad.The results were showed as follows:(1)optimization of preparation conditions for A.placenta protoplasts by orthogonal optimization and response surface methods,(2)Three cDNA libraries of different stages(conidia,germinated conidia,hyphum)were established and sequenced using the Illumina sequencing platform to figure out A.placenta genes differently expressed close to fungal development and pathogenicity.The findings were as follows.(1)0.7 mol/L NaCl as the optimal osmotic stabilizer,26.06 mg/mL enzyme,pH 6 and digesting time,digesting temperature and culture time were 4.03 h,30?and 64.6 h for protoplasts and production,followed with the yield of 4.41×107.The growth of protoplasts and spores of A.placenta were fully inhibited in the regeneration medium containing 30 mg/L and 120 mg/L herbicide(phosphinothricin,PPT)respectively.The result indicated that bar gene should be used as a selection marker gene.Plasmid PbarGPE containing bar gene was introduced into A.placenta protoplast.We selected putative transformants and characterized by PCR after 5 generations.(2)We obtained 18,557,966,15,738,191 and 25,861,449 clean reads from the RNA of conidia(CO),conidial germination(CG)and hyphum(HY)samples,respectively and Q30(those with a base quality greater than 30)and GC percentages were 92.59 and 55.40,83.56 and 55.88,92.46 and 55.23 respectively using the de novo sequencing.According to screening of DEGs,19 typical and very important genes closed to fungal development and pathogenicity were found in present study.The results based on RT-qPCR were following:6 genes including ao,wdr,hacl,cyp,ATP-syntH,and ?glu1 were up-regulated at the stage of CO;7 genes involed in pksp,nak1,zeb1,bcyp,npr,slps,and pep1 were also highly expressed at the stage of CG;6 genes,moreover,were highly expressed at the stages of CG and HY,especially at CG stage.We put forward the probable process of A.placenta developmental process including energy generation,signal transduction,protein synthesis,cell wall synthesis,cell division,cytoskeleton synthesis,secondary metabolistes like yellow pigment and tagatose.In respect of disease genes,we found that 2 kinds of genes involving hydrolase gene and immune resistance genes mainly existed.However,protease genes and chitinase genes were the uppermost hydrolase genes and mcl1 gene was the leading immune resistance genes.The first transformation system of this insect pathogenic fungus was established and optimized,meanwhile A.placenta DEGs of three developmental stages were firstly investigated.Our results also pave the way for the study on cloning,exprssion and functions of important genes through the following studies.