The study of chitin-associated proteins in Plutella xylostella

• Main Authors: Zhen-Hao Liao, 廖振皓
• Other Authors: Rong-Nan Huang
• Format: Others
• Language: zh-TW
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/5k6455

博士 === 國立中央大學 === 生命科學系 === 105 === Chitin is a major structural component of the cuticle and the peritrophic matrix (PM) in insects and is always associated with chitin binding proteins (CBPs) which are important for forming, maintaining and regulating the functions of these extracellular structures. Although the diamondback moth (DBM) Plutella xylostella, which has a high reproductive potential, short generation time, and characteristic adaptation to adverse environments, has become one of the most serious pests of cruciferous plants worldwide, the information on the chitin-containing structures and chitin turnover of the moth is presently limited. The overall objective of this dissertation was to study the PM proteins (PMPs), CBPs, and chitinases. Four trpsin like proteins which involved in immune or digestive functions were identified as PM associated proteins using 2-D electrophoresis and mass spectrometry. A genome-wide search for genes encoding proteins with ChtBD2-type (peritrophin A-type) chitin binding domains (CBDs) from P. xylostella was conducted. CBPs were classified, including 16 CPAP1s (cuticular proteins analogous to peritrophins with 1 CBD), 7 CPAP3s (CPAPs with 3CBDs), 12 PMPs and enzymes of chitin metabolism (7 chitinases and 7 chitin deacetylases) with a variable number of CBDs. The expression of CBP genes were evaluated by RT-PCR and the phylogenetic relationships among the CBPs from different order were further characterized. Moreover, we totally identified 15 chitinase genes using degenerated polymerase chain reaction (PCR) and rapid amplification of cDNA ends-PCR strategies coupled with searching chitinase-like sequences from the genomic and transcriptomic database. Based on the domain analysis of the deduced amino acid sequences and the phylogenetic analysis of the catalytic domain sequences, two of the gut-specific chitinases (PxCht25-1 and PxCht25-2) did not cluster with any of the known phylogenetic groups of chitinases and might be in a new group of the chitinase family. The expressions of PxCht5, PxCht7 and PxCht25-1 genes were performed in Escherichia coli, yeast and a baculovirus-insect cell expression system for enzymatic characterization. Their apparent molecular weights were different from the predicted ones, might be attributable to the incorrect folding or varying levels of glycosylation. Further biochemical analysis indicated that the recombinant proteins from the E. coli system lack endo-chitinase activity and baculovirus-insect cell might be the optimal chitinase expression system due to better kinetic values. With this information, a better understanding of the chitin-associated proteins involved in PM, cuticle and chitin turnover might help to develop novel chitin-targeted strategies for Lepidopteran pest control.