Identification And Expression Pattern Analysis Of Chitinase Genes From Paecilomyces Cicadae

Paecilomyces cicadae(Miquel)Samson,as one of the biocontrol fungi applied in China,is an entomopathogenic fungus on the larvae of Lepidoptera and Hemiptera insects.However,the basic research of this fungus is poorly reported,especially the pathogenic mechanism,which limits the application of this fungus on biological control of pests to some extent.In regard to this,the genome of P.cicadae ZJ1611 was sequenced,assembled and annotated.On a genomic scale,the virulence genes of P.cicadae were screened.Eighteen P.cicadae chitinase genes were identified,and the classification,domain and expression patterns of these genes were also analyzed.The results of this study provided important information for further exploring the pathogenic mechanism of P.cicadae,which will help to improve its control effect and application potential.The main findings are as follows:(1)The draft genome of P.cicadae contained 1218 scaffolds with G+C content of 53.11%.The genome size was about 33.8 Mb,including 11106 genes.Compared with other insect pathogens,the genome size of P.cicadae is similar to that of Cordyceps militaris and Beauveria bassiana,but smaller than the size of the Metarhizium species(such as M.robertsii,M anisopliae and M.acridum).(2)Basing on the virulence genes characterized from Beauveria and Metarhizium species,we screened 464 virulence genes from 41 protein families in the P.cicadae genome.Sequence analysis of selected 16 virulence genes of P.cicadae showed that the molecular weight range is 10.58-224.95 KDa,and PChyd being the smallest,PCacy being the largest.The isoelectric points of these genes were less than 7,except for PCmpll,PCabc,PCmfs,and PCpta.The results of expression patterns showed that the relative expression levels of PCmpll,PCcyp450,PCpr1b,PCmsn,PCabc,PCmfs and PCacy in AP sample were significantly higher than those in other samples,and PCdtx and PCchi in PDA and BS were significantly higher than in other samples,respectively.We also found that the potential functions of most these genes were similar to those virulence genes described in Beauveria and Metarhizium,except for PCmsn,PCabc,PCdtx,PCmfs and PCacy.Among them,PCabc and PCmfs may be have rich functions in the pathogenic development of P.cicadae.(3)Eighteen putative glycoside hydrolase family(GH)18 chitinase genes were identified in P.cicadae genome.The conserved domains,DxxDxDxE,S/AxGG,CBM1,CBM 18,and CBM50 were also detected in P.cicadae chitinases.Based on the phylogenetic analysis,the 18 predicted chitinases were classified into subgroups A(7 chitinases),B(8 chitinases)and C(3 chitinases).Analysis of the exon-intron organizations show that most of these chitinases contained intron.The results of expression patterns showed that four genes(PCchiA4,A6,B1 and B4)had high transcript levels in the nine different samples.We also found subgroup A chitinases presented higher transcript levels compared to all other chitinase transcripts.Meanwhile,all chitinase genes were ubiquitously expressed in chitin condition,suggesting that they might play an important role in chitin degradation.In addition,PCchiBl chitinase presented higher transcript level compared to all other chitinase transcripts,suggesting that PCchiBl might be a vital P.cicadae chitinases and participate in some important physiological pathways.(4)The sensitivity test of P.cicadae for hygromycin B(HPH)and glyphosate(PMG)were detected.We also constructed the system of protoplast transformation for P.cicadae to knockout the sgA genes.A 1384bp fragment of HPH was amplified from the positive transformants.