Studies On The Expression And Function Of Insulin Signaling Pathway Genes From Coridius Chinensis

Cordius chinensis belongs to Hemiptera: dinidoridae,which is a resource insect with development potential.It was found that C.chinensis is rich in nutrients,such as fat and protein.In addition,its extracts also have broad-spectrum antibacterial activity and anticancer effect.C.chinensis is the food material which can be used as medicine and food in China.Compared with the natural environment,the nymphs are cultured can’t reproduce normally,which brings huge challenge to the formalization cultivation of C.chinensis.The insulin signaling pathway of insects can regulate important physiological processes,such as the growth,reproduction and metabolism.At present,there are few researches about the reproduction of C.chinensis.In order to provide a theoretical basis for exploring the cultivation technology of C.chinensis,our reasearch focuses on the molecular mechanism of the insulin signaling pathway for adjusting the reproduction of the C.chinensis.In this study,the Open Reading Frame of insulin signal pathway genes were characterized by Reverse transcription-polymerase chain reaction(RT-PCR)based on the transcriptome of C.chinensis.These key factors include Insulin receptor(INR),Phosphatidylinositol-3-kinase(PI3K),Phosphoinositide-dependent kinase(PDK),Protein kinase B(AKT/PKB)and Forkhead box protein O(FOXO).Bioinformatics softwares were used to analyze the sequence characteristics and conservativeness of these genes.We analyzed the expression patterns of insulin signaling pathway genes at different developmental stages and in various adult tissues by using real-time quantitative PCR(RT-q PCR).Disruption of the Insulin receptor and Forkhead box protein O effected the ovarian development in C.chinensis,respectively.Cc FOXO was successfully expressed by Escherichia coli BL21(DE3).The detailed results are as follows:1、 Characteristic analysis of insulin signal pathway genes from C.chinensisThe nucleotide sequences of CcINR,CcPI3 Kc,CcPI3 Kr,CcPDK,CcAKT and Cc FOXO were identified from the full-length transcriptome of C.chinensis and verified by RT-PCR and Sanger sequencing.Then bioinformatics analysis indicated that the open reading frame length of the six genes was 3828 bp,2589 bp,2817 bp,1461 bp,1563 bp and 1152 bp,respectively,encoding 1275,862,937,486,520 and383 amino acids.Multiple sequence alignments indicated that the domains of six genes were conserved and the insect insulin signaling pathway was conserved in the evolutionary process relatively.The phylogenetic tree elucidated that C.chinensis and Hemiptera clustered into a branch,among them,C.chinensis has the closest homologue with Halyomorpha Halys.2、Spatiotemporal expression profile with insulin signaling pathway genes of C.chinensisThe expression patterns of insulin signaling pathway genes from C.chinensis at various developmental stages,include eggs,nymphs and adults were analyzed by RTq PCR.The expression levels of six genes remained at high levels in the egg stage,and it is speculated that insulin signaling pathway is related to embryonic development.The expression levels of Cc PI3 Kc,Cc PI3 Kr,Cc PDK and Cc AKT in fourth-instar nymph were higher than those in other nymphs,which suggesting that insulin signaling pathway maybe involved in the regulation of wings.The tissues specific expression results showed that six insulin signal pathway genes were highly expressed in testis and ovary,on the side,the expression levels of Cc PI3 Kr,Cc PDK,Cc AKT and Cc FOXO genes in midgut and fat body were also significantly higher than those in other tissues.It is speculated that the insulin signal pathway maybe involved in the regulation of reproduction,digestion and absorption,nutrient storage and energy metabolism of C.chinensis.Insulin signal pathway genes were specifically expressed at different developmental stages and in various adults tissues,it is indicats that they play an indispensable role in the growth and development of C.chinensis.3、Functional study of CcINR and CcFOXO genes on ovarian development in C.chinensisThe injection of dsRNA successfully silenced the CcINR gene of C.chinensis,compared with the control,the silencing efficiency reached 42.73%,77.34%,and30.83% at 48 h,72 h,and 96 h,respectively,and the transcription levels of Cc Vg and Cc Vg R decreased by 59.93% and 50.79%,respectively.The length of the ovarian tube was significantly shorter than the control group.The expression levels of Cc FOXO significantly decreased by 34.93%,65.06%,and 13.09% at 48 h,72 h,and 96 h,respectively,after ds Cc FOXO injection.Meanwhile the expression of CcVg and CcVgR reduced by 87.36% and 84.48%,respectively.The length of the ovarian tube was significantly shorter than that of the control group.It is suggested that Cc INR and Cc FOXO play an indispensable role in the ovary development of C.chinensis.4、 Prokaryotic expression and analyze of Forkhead box protein O from C.chinensisThe nucleotide sequence of CcFOXO was optimized according to the preference of codon usage in E.coli.The restriction endonuclease recognition sites for Nde I and Xho I were added to the 5’ end of the coding region of Cc FOXO,respectively,then subcloned into p ET-28b(+)vector.The recombinant plasmid,p ET-28b(+)-FOXO,was transformed into E.coli BL21(DE3).The positive clones were cultured at 37℃overnight,and the expression of the target protein was induced by IPTG.The target protein was purified from the inclusion bodies by using the 6×His tag and Ni-NTA resin for polyclonal antibody preparation.