| 摘要: | 小菜蛾 (Plutella xylostella, diamondback)，繁殖能力強，生長世代短，可適應各種惡劣的環境，為世界性的十字花科作物重要害蟲，然而其幾丁質相關構造及酵素目前仍未被詳細研究。幾丁質為構成昆蟲表皮及圍食膜 (peritrophic matrix: PM) 之重要物質；幾丁質通常會與幾丁質結合蛋白 (chitin binding proteins: CBPs) 結合，CBPs對於幾丁質重要構造的形成、結構維持以及功能的調控，扮演重要的角色。本研究解析小菜蛾體內幾丁質重要構造之蛋白與幾丁質相關酵素，特別針對圍食膜蛋白 (PM proteins: PMP)、CBPs以及幾丁質酶 (Chitinase: Chts) 等幾丁質相關蛋白，根據CBPs之基因表現情形、親緣關係分析及幾丁質酶選殖與表現定性，期望獲得有助於研發新式鱗翅目害蟲防治之重要資訊。利用2-D電泳與質譜分析，可鑑定到四個 PM 相關蛋白，其身分皆為胰蛋白酶，分別負責免疫與消化之功能。另外利用基因體搜尋CBPs (ChtBD2-type or peritrophin A-type)，共找到16個具有一個幾丁質結合區域的表皮蛋白 CPAP1s (cuticular proteins analogous to peritrophins with 1 chitin binding domain (CBD))、7個具有三個幾丁質結合區域的表皮蛋白 CPAP3s (CPAPs with 3CBDs)、12個PMPs，以及幾丁質相關酵素，包括7個Chts和7個幾丁質脫乙醯酶 (chitin deacetylases: CDAs)。最後利用基因選殖技術 (rapid amplification of cDNA ends-PCR)，搭配基因體與轉錄體之搜尋，總共可找到15個Chts，其中本論文選殖的PxCht25-1 和 PxCht25-2，為小菜蛾特有之新式Chts，且只在中腸特有表現。另外將選殖而來的PxCht5、PxCht7 和PxCht25-1，分別於大腸桿菌、酵母菌與昆蟲細胞表現蛋白。大腸桿菌表現系統之重組蛋白，可能因蛋白摺疊錯誤，影響了結構，使其喪失幾丁質內切之能力；酵母菌表現系統之重組蛋白，則因過度醣化修飾，使得分子量遠大於預期，且影響了活性；昆蟲細胞表現系統則為幾丁質酶最佳表現系統，擁有最佳的酵素動力學數值。;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. |
