Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 9/29/2004
Publication Date: 1/3/2005
Citation: Zhu, Y., Liu, X., Maddur, A., Oppert, B.S., Chen, M. 2005. Cloning and characterization of chymotrypsin- and trypsin-like cDNAs from the gut of the Hessian fly (Mayetiola destructor (say)). Insect Biochemistry and Molecular Biology 35: 23-32. Interpretive Summary: Chymotrypsins and trypsins in the insect digestive system are proteinase enzymes that break proteins ingested from food into smaller pieces before these proteins can be absorbed and utilized as nutrients. Accordingly, inhibitors to chymotrypsins or trypsins are targets for bioengineering to generate insect resistant plants. This manuscript reports the cloning and characterization of several genes that produce chymotrypsins or trypsins from the Hessian fly, one of the most destructive insect pests of wheat. This research provides a foundation for further studies that may lead to the development of resistant wheat using chymotrypsin or trypsin inhibitors.
Technical Abstract: Fifteen unique cDNA clones encoding trypsin- or chymotrypsin-like proteins were cloned and characterized from a gut cDNA library derived from Hessian fly [Mayetiola destructor (Say)] larvae. Based on sequence similarities and phylogenetic distances, the cDNAs were sorted into five gene groups which were named MDP1 to MDP5, respectively. Two of the gene groups, MDP1 and MDP2, encoded chymotrypsin-like proteins while the other three encoded putative trypsins. All deduced proteins have conserved His87, Asp136, and Ser241 residues for the catalytic triad and three pairs of cysteine residues for disulfide bridge configurations. The substrate specificity determination residue at position 235 was also conserved in the putative trypsins and chymotrypsins. In addition, all the deduced protein precursors had a typical secretion signal peptide and activation peptide. Northern blot analysis revealed that all these gene groups were exclusively expressed in the larval stage. The expression profiles for each gene group varied significantly in different ages of the larva as well as in different tissues. Protease activity analysis of gut extract using specific inhibitors demonstrated that serine proteases were the major digestive enzymes in the gut of M. destructor larvae. Serine protease inhibitors inhibited as high as 90% proteolytic activities of gut extract, while inhibitors specific to other proteases including cysteine proteases, aspartic proteases, and metallo-proteases inhibited only 10 to 24% of gut protease activity.