CDNA Cloning And Characterization Research Of A Serine Protease In The Midgut Of Heliothis Viriplaca

Proteases are mainly involved in protein synthesis and degradation, and they are essential enzymes to maintain growth and development for all organisms. Serine protease is an important member of proteases and important proteolytic enzymes, in which serine is the active center. Research shows that in insects, serine protease is involved in several important physiological processes, such as digestion, growth, innate immune reponse and tissue reconstruction. In insects, because serine protease plays an important role in digestion and innate immune, it has become a hot research point in domestic and foreign laboratories. At present, serine protease is researched mainly in model insects, such as Drosophila melanogaster and Bombyx mori, while little study has been done on important agricultural pests. Heliothis viriplaca is an important defoliator to soybean, it has become one of the main soybean pests.In this study, the serine protease cDNA sequence from H. viriplaca was named HvSP, which was deposited in Gen Bank with the accession number KT907053. The cDNA sequence was expressed in E. coli expression system and the expressed fusion protein was denatured, purified and renatured. The activity of recombinant protein was determined with BTEE as the substrate. At the same time, HvSP expressed on mRNA level in different tissues was studied. Finally, the effect of RNA interference on HvSP was detected by injecting double-stranded RNA in vitro. The main results are summarized as follows:(1) The full-length cDNA sequence of HvSP was 1017 bp which included an open reading frame of 886 bp, encoding 295 amino acids, containing 34 bp and 95 bp of 5′ and 3′ noncoding region and a signal peptide containing 17 amino acids in N-terminal. The molecular weight was about 30.8 k Da, and the isoelectric point was 8.27. The identities of HvSP protein and those from other lepidopteran insects were 46%~92% by NCBI Blast. NCBI Blast found that the high homology of HvSP protein were chymotrypsin, therefore we thought HvSP was chymotrypsin in this research.(2) HvSP was cloned into prokaryotic expression vector p ET21 b and expressed in BL21 cells. After IPTG induction, SDS-PAGE was used to analyze the expressed protein. The result shows there is an obvious target protein band which was close to 30.8 k Da. This was similar with predict protein molecular weight, while target band was not detected in the empty vector of p ET21 b. T he expressed fusion protein was denatured, purified and renatured. The activity of recombinant protein was determined with BTEE as the substrate. The enzyme activity determination indicated that in Tris-HCl buffer solution of 0.1 mol/L, the recombinant protein activity was 28.7 U/mL when p H was 8.5.(3) The results of real-time quantitative PCR showed that HvSP expressed on mRNA level in different tissues, especially in the midgut, but not expressed in the salivary gland. Compared with other tissues, HvSP gene was expressed the highest in the midgut and significant difference with other tissues(p<0.05), the expression of HvSP in midgut was 14-fold than in fat bodies. This indicated that HvSP gene mainly existed in midgut of H. viriplaca.(4) The RNAi research showed that, after 72 h, HvSP gene expression of H. viriplaca injected with 2 μL ds HvSP at the concentration of 2 μg/μL decreased by 65%. The results showed that efficiency of RNAi-mediated knockdown of HvSP gene was successful.