Identification Of M14 Family Metal Carboxypeptidases In Antheraea Pernyi And Transcriptional Response Of Digestive Enzymes Triggered By Starvation

The Chinese oak silkworm,Antheraea pernyi,usually feeds on Quercus leaves in the field.Due to its high contents of protein and unsaturated fatty acid,A.pernyi is regarded as an edible insect.A.pernyi cannot directly absorb dietary proteins,which must first be enzymatically digested in the gut and then absorbed.Thereby,the conversion efficiency of the dietary protein into silkworm protein affects the protein content of A.penryi.Generally,endopeptidases first degrade dietary proteins into peptides,which will be further hydrolyzed into free amino acids by exopeptidases and absorbed for growth and development.Many studies have showed that M14 family metal carboxypeptidases（MCPs）participate in the conversion of external proteins into internal proteins and play some roles in the digestion and pathogenic invasion in the gut of insects.However,MCPs in A.pernyi remain to be analyzed.Therefore,based on the highly-expressed ApMCP1 in the gut,members of the M14 family metal carboxypeptidases were first identified in A.pernyi genome.And then the expression patterns of MCP genes,which are expressed in the gut,after starvation and immune were studied.Furthermore,the genes of digestive enzymes were identified by comparative transcriptome analysis between the guts of starvation and refeeding.All these efforts pay a foundation for studying the digestive function of these genes and improving the conversion efficiency of dietary protein.The main results of this study are as follows:1.The highly-expressed M14 metal carboxypeptidase gene（ApMCP1）in the gut of A.pernyi was successfully identified using comparative transcriptome analysis.After that,the full-length ORF of ApMCP1 was cloned,showing that the length of open reading frame was 1,194 bp,which encoded a 397-amino acid protein with a predicted molecular weight of 45 k Da.Multiple sequence alignment between ApMCP1 and its homologous proteins revealed that the key amino acid residues in the Zn＿pept domain were highly conserved.2.Used ApMCP1 as the query,14 members of MCPs（ApMCP1–ApMCP14）were identified in A.pernyi genome.All of them have typical Zn＿pept domains and two Znanchoring motifs of M14 family,and were further classified into M14 A,M14B,and M14 D subfamilies using phylogenetic analysis.3.Spatial and temporal expression analysis showed that ApMCP1 and ApMCP9 were mainly expressed in the gut,and would be used as the representatives for functional study of MCPs.4.The dissections made following starvation for different hours showed that gut contents were running down and digested for 48 h.Refeeding resumed the condition of the gut content.In addition,ApMCP1 and ApMCP9 displayed opposite expression patterns after starvation and refeeding,highlighting their functional divergence during digestion.Following natural infection with baculovirus NPV,their expressions were significantly upregulated in the gut of A.pernyi,indicating that they participated in the immune reponse on pathogenic infection.5.Based on comparative transcriptome analysis between starvation and refeeding,59 digestive enzymes were identified in differentially expressed genes（DEGs）,including 17 DEGs after starvation,37 DEGs after refeeding,and 3 DEGs shared between starvation and refeeding.All the differentially-expressed digestive enzymes were annotated as serine proteases,trypsins,chymotrypsins,metalloproteinases,cysteine proteases,aspartic proteases,or lipases.Two genes of autophagy related proteins were also found.All these results have paved an important way for studying the physiological function of digestive enzymes in the gut of A.pernyi.In summary,these novel gut-expressed M14 metal carboxypeptidase and other digestive enzyme genes in A.pernyi provide an important starting point for raising the conversion efficiency of dietary protein and breeding special variety with higher nutritional value for consumption.