| Corn is one of the three major cereal crops in the world, and it plays a decisive role on food, fodder and industrial processing as raw materials. In recent years, with the expansion of corn acreage and continuous cropping year by year, the damage of corn borer Ostrinia furnacalis on corn production was aggravated gradually, which seriously impacted corn yield. Insect-resistant transgenic corn is developing in China now. Serine proteinase inhibitors (Serpins) are broadly distributed family of protease inhibitors, and most members of this family have an specific inhibitory activity against serine proteases. So far, more than 1500 types of Serpins had been found in animals, viruses, plants, bacteria and archaea. Serpins generally contain 350～400 amino acid residues, and the molecular weight is about 40-100 kDa. The conserved three dimensional structure of Serpins also contains 8～9 alpha-helices and 3 beta-sheets. Serpins have a exposed amino acid region named reactive centre loop (RCL) around its reactive site at the C terminus. This region which can be covalently bound to serine proteases formed a surface loop protruding from the body of the molecule, and then Serpin itself is cleaved at PI site. The amino acids of P1 may play an important role in the binding process of Serpins.In this paper, four P1 site mutants of candidate transgenic target gene, serine protease inhibitor (Serpin 1) which is related to insect immunity were constructed, and four kinds of mutant proteins were expressed for the functional analysis, respectively. Serpinl had been cloned from O. furnacalis in our lab, and the full length cDNA of the Serpinl was 1336 bp (accession number:JX524148), containing a 1188 bp open reading frame (ORF), a 36-bp 5-untranslated region, and a 112-bp 3-untranslated region with a poly(A) signal. The open reading frame (ORF) encodes a 395-residue protein with a theoretical molecular mass of 43.3 kDa and an isoelectric point of 4.92. O. furnacalis Serpin1 (Of-Serpin1) contains a putative signal peptide followed by a conserved domain including a reactive center loop (RCL) with a hinge region (E344 to S353) and a predicted P1-P1’ cleavage site (Leu360-Ser361). In order to investigate the role of P1 site in Of-Serpin1, mutation primers were designed, and the wild-type serpinl PI sites of leucine L360 (CTA) was mutated to arginine R360 (AGG), lysine K360 (AAG), phenylalanine F360 (TTC) and serine S360 (AGT), respectively, and the DYKDDDDK (FLAG tag) was added at the Of-Serpin1 N-terminal. The Of-Serpin 1 ORF of wild type and four kinds of mutants were inserted intopET-28b vector, and transformed into Escherichia coil BL21 strain. The fusion protein of Of-Serpins were purified with Ni2+ cheated affinity chromatography and then identified by Western blotting. Inhibition kinetics of the wild type and four mutants Of-Serpinl (R, K, F, S) recombinant protein were analyzed. The results showed that the values of Vmax and Km of chymotrypsin were lowest among four mutants after the addition Of-Serpinl S360 (AGT), and the expressed Of-Serpinl (P1, S360) showed the greatest affinity ability with chymotrypsin, and the affinity ability among four Of-Serpinl P1 site mutants was S360> K360> F360> L360> R360 in turn. The Ki value of Of Serpin1 S360 (AGT) is the lowest after the addition of chymotrypsin, and the expressed Of-Serpinl (P1, S360) showed the strongest inhibition with chymotrypsin, and the inhibition ability of Of-Serpinl P1 sites mutants were S360> K360>F360> L360> R360 in turn. Bioinformatics analysis indicated that the Of-Serpinl was an anti-chymotrypsin like other inhibitor, but the results of selective inhibition experiment showed that both bovine pancreatic trypsin and bovine pancreatic chymotrypsin can cleave the recombinant wild-type and mutant Of-Serpin1. |
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