Cloning And Functional Analysis Of Two Cathepsin Genes From Asian Corn Borer

The Asian corn borer(Ostrinia furnacalis Guenée,Pyralidae,Lepioloptera)is a major pest in maize production in China.It belongs to the completely metamorphosed insects.In the process of embryonic and metamorphosis development of such insects,cathepsin plays an important role in the process of obvious periodic changes in its morphology and structure.In this paper,two cathepsin genes of O.furnacalis were cloned and analyzed by bioinformatics.At the same time,the expression patterns of different developmental stages and different tissues of larvae were analyzed.The antigen was obtained by prokaryotic expression,polyclonal antibody was synthesized,and the effects of two proteases on the metamorphosis development and fecundity of the two proteases in Asian corn borer were studied based on RNAi and Western blot technology.The main results are as follows:1.Cloning and identification of two cathepsin genes in O.furnacalisThe differentially expressed cathepsin gene was screened by Transcriptome data,cloned and verified by RT-PCR technology and compared online by NCBI Blast,After cloning and NCBI comparison verification,it was determined that the sequences were Of CTSB and Of CTSL(Gen Bank: ON204608.1;OL744079),and the ORF Finder prediction results showed that the open reading frame(ORF)length was 1014 bp and1026 bp,the amino acid sequence length of the encoded protein was 337 aa and 341 aa,and the relative molecular weight of the encoded protein was 37.3 k D and 38.2 k D,respectively.Biological information analysis showed that two cathepsin genes had signal peptides,and belonged to hydrophilic proteins.Phylogenetic analysis showed that the amino acid sequences of the two cathepsins of O.furnacalis and other insects had different degrees of similarity,and were most closely related to Galleria mellonella.2.The temporal and spatial expression patterns of two cathepsin genes in O.furnacalisThe expression profiles of two cathepsin genes in O.furnacalis showed that both cathepsin genes were highly expressed in the egg and pupal stages of O.furnacalis.It is speculated that it may be involved in the process of tissue reconstruction during the embryonic development and pupal stage of O.furnacalis.In different tissues,the relative expression of Of CTSB in silk gland and epidermis was the highest,which was17.59 times and 17.28 times of that in hindgut,respectively.Of CTSL was highly expressed in hemolymph,followed by malpighian tubule,epidermis and fat body,indicating that two cathepsin genes may be involved in physiological processes such as insect molting,silking and immunity.3.Prokaryotic expression of cathepsin B and preparation of polyclonal antibodyAfter codon optimization of the nucleic acid sequence of Of CTSB,the nucleic acid sequence of the signal peptide region was removed,and the specific bands and imprints of the target protein appeared around 35 k Da at 20 °C and 37 °C induced,inducing at0.5 mmol/L IPTG at 20°C and 37 °C culture,and detected by SDS-PAGE and Western blot,and the expression of the target protein was greater at 20 °C.Therefore,the recombinant protein was purified from the inclusion body by 6 × His tag and Ni-NTA resin using 0.5 mmol/L IPTG,overnight culture at 20 °C as a condition for large-scale expression.The synthesized polyclonal antibody was subjected to ELISA titer detection and Western blotting analysis.The synthesized polyclonal antibody had a titer greater than 1:256 000 and could bind specifically with the initial antigen to obtain accurate CTSB polyclonal antibodies.4.Functional study of cathepsin B and L in O.furnacalis In order to explore the effects of cathepsin B and L on the metamorphosis development and reproduction of Asian corn borer.In this study,the ds RNA of the target gene was injected into the nanocarrier to successfully silence the two cathepsin genes of the Asian corn borer.The results showed that the silencing efficiency of the two cathepsin genes reached a significant level.The expression levels of Of CTSB in the 4th and 5th instar larvae were down-regulated by 45.30 % and 52.72 %,respectively,and Of CTSL was65.22 % and 31.55 %.Silencing of Of CTSB and Of CTSL can lead to abnormal molting and even death of larvae.In the larval to pupal stage,it will produce abnormal pupae and affect its metamorphosis.The interference efficiency of the two cathepsin genes in adult stage was 40.31 % and 35.32 %,respectively.After silencing Of CTSB and Of CTSL,the average total number of eggs laid per female adult decreased by 66 and235,respectively.The results showed that Of CTSB and Of CTSL were involved in the molting,pupation and reproduction of O.furnacalis,and played an important role in insect growth and reproduction.