The Full-length CDNA Sequence Cloning And Expression Spectrum Analysis Of The Argoanute Protein Family In The Gampsocleis Gratiosa

The discovery of RNA silencing has been known for two dacades.So far RNAi has widely used for insect gene function research.The Argonaute protein family,which is the core molecule of RNAi,has also received intensive attention.The Argonaute protein family has a large members and varies in number from species to species.The Argonaute protein family members usually contain four domains: variable N-terminus,PAZ,MID and PIWI,among which PAZ and PIWI belong to the conservative domain and also are iconic functional domains of Argonaute protein family.Although the composition of the Argonaute protein family in some insects has long been known,such as flies,silkworms,and schistosomiasis,the exact composition of the Argonaute protein family in most insects remains unclear.In this study,based on the high-throughput transcriptome data obtained before,RT-PCR,RACE,and bioinformatics techniques were used to identify,clone,and analyze Argonaute protein family members.And then using real-time fluorescence quantitative PCR technique to analyze the level of transcription in different developmental stages and different tissues.1 Identification and cloning of Argonaute protein family in G.gratiosa.By comparing the cDNA sequences in G.gratiosa,Drosophila melanogaster,Locusta migratoria and Bombyx mori,four members(AGO1,AGO2,AGO3 and PIWI)has been identified.The full-length cDNA sequences were AGO1 2965 bp,AGO2 3946 bp,AGO3 3244 bp,and PIWI 3462 bp,respectively.By bioinformatics analysis the ORFs of four genes encoded 834,1101,912,and 913 amino acid residues respectively,and their predicted molecular masses were 94.61 kDa,123.91 kDa,102.57 kDa,and 102.68 kDa,respectively.All four members were basic proteins,and their isoelectric point(p I)were 9.31,9.66,9.31,and 9.55,respectively.We found two typical conserved domains(PAZ and PIWI)by predicting the protein structure via SMART software.and there were DDH catalytic triad in the PIWI domain of all members.Phylogenetic analysis of AGO1,AGO2,AGO3,PIWI in G.gratiosa and other insects revealed that AGO and PIWI subfamilies clustered into independent branches,respectively.2 Expression profile analysis of G.gratiosa Argonaute protein familyIn order to conduct a preliminary study on the potential functions,the Argonaute protein family expression profile in different tissues and different developmental stages of males and females was analyzed by real-time fluorescence quantitative PCR.These four genes were expressed in various stages of development,and the ago1 and ago2 expression patterns were similar.However,the expression levels of ago1 and ago2 at the stages of young larva were significantly lower than those at the stage of egg.This indicates that ago1 and ago2 not only play an important role in the regulation of the growth stage,but may even have essential significance in the maturation and development of oocytes in G.gratiosa.In different tissues of male and female adults,ago1 and ago2 are highly expressed in the midgut,hindgut and gastric cecum.Combined with their known functional effects,it is not difficult to infer that the two genes have a great influence on regulating biological metabolism and preventing virus invasion.At the same time,there are two periods of high expression of the two genes,one is the period of metamorphosis from egg to larva,and the other is the period of emergence from larva to adult.So we have another hypothesis,is it possible that the ago1 and ago2 are involved in the accumulation of related proteins in G.gratiosa metamorphosis and feathering,and this surmise needs further study.The piwi and ago3 are highly expressed in egg instar,7th instar,and adult instar of male and in 4th instar of female.It is speculated that the piwi and ago3 may play an important role in the initiation and maturation of the male germ cells,and may be more inclined to play a role in the maintenance of female germ cells.Moreover,piwi and ago3 are not only highly expressed in the gonads,but also expressed in other tissues more or less.It is speculated that piwi and ago3 may have functions alone that do not need to be combined with piRNA.