Cloning And Function Identification Of Serine Protease CDNAs In The Midgut Of Mythimna Separata(Lepidoptera:Noctuidae)

Serine proteases(SPs), mainly including trypsin(EC number: 3.4.21.4) and chymotrypsin(EC number: 3.4.21.1), are a diverse group of important proteolytic enzymes identi?ed by their Ser residue at the active site. Serine proteases are the main digestive enzymes in lepidopteran insects,which can contribute up to 95% of the total digestive activity in the lepidopteran larval gut. And they play numerous roles in insect growth and development, such as food protein digestion,molting process and immune response. In addition, some reports demonstrated that serine proteases in the midgut of lepidopteran larval are involved in the process of Bacillus thuringiensis(Bt)protoxin activation and toxin degradation, pest’s resistance to Bt toxins may depend on the changes of serine proteases in the expression level or type(such as trypsin and chymotrypsin). So serine proteases in the lepidopteran larval gut have been the focus of research, gradually becoming the target of pest control, and provide a direction for the development of a new generation of insecticides.The oriental armyworm, Mythimna separata, is one of the major pests of crops in Asian countries. In order to explore the roles of serine proteases in Mythimna separata, two cDNA sequences of a trypsin-like and a chymotrypsin-like serine protease were obtained from the midgut of Mythimna separata larva and they were named as MsT(GenBank ID: KP730443) and MsCT(GenBank ID: KP730444), respectively. Then the MsT and Ms CT c DNA sequences were expressed in Escherichia coli expression system and the activity of recombinant proteins were determined.Meanwhile, the transcriptional level of MsT and MsCT genes in different tissues and developmental stages were studied. And the transcriptional response of MsT and MsCT genes to starvation,re-feeding, 20-hydroxyecdysone(20E) and Cry1 Ac protoxin were also studied. Lastly,RNAi-mediated knockdown of MsT and MsCT genes were carried out by means of microinjection of a dsRNA to further investigate the consequences of these genes. The main research findings are as follows:1. The full-length cDNA sequence of MsT is 856 bp in length with an open reading frame of765 bp, encoding 255 amino acid residues with the predicted molecular weight of 27.1 kDa and pI of 9.04. Multiple sequence alignment indicated that MsT shares 50%-84% amino acid sequence identities with serine proteases from other lepidopteran insects, and shares the highest amino acid sequence identity(84%) with trypsin-like serine protease from Mamestra configurata(GenBank2.accession no.: ACR15970).2. The full-length cDNA sequence of MsCT is 971 bp in length with an open reading frame of891 bp, encoding 297 amino acid residues with the predicted molecular weight of 31.0 kDa and pI of 8.63. Multiple sequence alignment indicated that MsT shares 44%-84% amino acid sequence identities with serine proteases from other lepidopteran insects, and shares the highest amino acid sequence identity(84%) with chymotrypsin-like serine protease from M. configurata(GenBank accession no.: ACR15972).3. The recombinant vector MsT-pET21 b and MsCT-pET21 b were expressed in E.coli BL21 prokaryotic expression system, respectively. Then the recombinant proteins were determined as the target proteins using SDS-PAGE and Western-blot analysis. It was indicated that the recombinant proteins were inclusion body protein through protein solubility analysis. After the recombinant protein was treated with denaturation, purification and renaturation processes, the activity of renatured recombinant protein was determined with BApNA as the substrate. The results showed that, in Tris-HCl buffer solution, the activity of recombinant protein increased along with the increase of pH, which reached 23.24 U/mL when the pH was 9.5; In Glycine-NaOH buffer solution,the activity of recombinant protein firstly increased and then decreased with the variation of p H, which reached the highest activity(35.74 U/ mL) when the pH was 10.0.4. The transcriptional level of MsT and MsCT genes in different tissues and developmental stages were defined. The results showed that MsT and MsCT genes transcripts were both detected in the foregut, midgut, Malpighian tubules, fat body and peritrophic membrane. Particularly, it was in the peritrophic membrane that the transcripts both reached the highest levels. And MsT and MsCT genes transcripts were both mainly detected at the 6th instar larvae, prepupal and pupal stage. The MsT transcript reached the highest level at the pupal stage, while the MsCT transcript levels were almost similar at 6th instar larvae, prepupal and pupal stage.5. The transcriptional response of MsT and MsCT genes to starvation and re-feeding were defined. The results showed that the expressions of both MsT and MsCT were significantly down-regulated after 24 h starvation and up-regulated again by re-feeding. It indicated that MsT and MsCT genes were most likely involved in food protein digestion in M. separata.6. The transcriptional response of MsT and Ms CT genes to 20 E were defined. The results showed that the expression of MsT was up-regulated in 6th larvae injected with 20 E at the concentrations of 2 ug/larva after 12 h over the control levels, which reached the highest level after24 h injection; however, the expression of MsCT in response to 20 E was down-regulated after 24 h injection. It indicated that MsT gene was most likely involved in molting process in M. separata.7. The transcriptional response of MsT and MsCT genes to Cry1 Ac protoxin were defined. The results showed MsT transcripts was not significantly affected by Cry1 Ac protoxin over the control levels; however, MsCT transcripts was significantly up-regulated after 24 h when 3thlarvae were fed on corn leaves with Cry1 Ac protoxin. It indicated that Ms CT gene was most likely involved in Bt protoxin activation in M. separata.8. The RNAi research of MsT and MsCT genes showed that, after the M. Separata 4th larvae was injected with dsMsT and dsMsCT at the concentration of 4 ug/larva, the transcriptional levels of MsT and MsCT genes decreased 76.69% and 86.16% respectively after 12 h, and the RNAi-mediated knockdown efficiency of the MsT and MsCT genes were still alive after 48 h.Meanwhile, The mortality of M. Separata 4th larvae after injecting single dsRNA of ds MsT and dsMsCT were 26.66% and 32.22%, respectively.