博碩士論文 952404003 詳細資訊




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姓名 廖振皓(Zhen-Hao Liao)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 小菜蛾幾丁質相關蛋白之研究
(The study of chitin-associated proteins in Plutella xylostella)
檔案 [Endnote RIS 格式]    [Bibtex 格式]    至系統瀏覽論文 (2020-1-1以後開放)
摘要(中) 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? (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?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?,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? (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?最佳表現系統,擁有最佳的酵素動力學數值。
摘要(英) 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.
關鍵字(中) ★ 小菜蛾
★ 幾丁質
關鍵字(英)
論文目次 摘要.............i
摘要.............i
Abstract....... ii
致謝............iv
目錄............v
圖目錄.... viii
表目錄.... x
縮寫表... xi
第一章 緒論...... 1
1.1 昆蟲幾丁質結構與組成.... 1
1.2 昆蟲幾丁質動態......... 1
1.3 幾丁質構築昆蟲重要構造 ........ 2
1.4 幾丁質結合蛋白..................3
1.5 幾丁質為蟲害管理之標的........4
1.6 研究目的............. 5
第二章 材料與方法 ................7
2.1 小菜蛾之飼養 ................7
2.2 分離與鑑定圍食膜蛋白 ........7
2.2.1 圍食膜蛋白萃取......... 7
2.2.2 二維蛋白質凝膠電泳 (Two-dimensional electrophoresis, 2-DE).................... 7
2.2.3 膠體內分解胜?片段 (In-gel tryptic digestion).... 8
2.2.4 胺基酸序列分析及蛋白質資料庫搜尋....... 8

2.3 小菜蛾幾丁質結合蛋白之研究 ........................... 9
2.3.1 基因體資料庫搜尋 ............................ 9
2.3.2 幾丁質結合蛋白基因表現情形 ........................9
2.3.3 序列及演化樹分析 ............................ 10
2.4 小菜蛾幾丁質?之研究 ............................. 10
2.4.1 基因體資料庫搜尋 ............................ 10
2.4.2 幾丁質?之基因選殖 ............................ 10
2.4.3 幾丁質?基因表現情形 .......................... 11
2.4.4 幾丁質?表現載體之建構 ......................... 11
2.4.5 純化幾丁質?重組蛋白 ........................... 12
2.4.6 膠體電泳及西方墨點法 .......................... 13
2.4.7 幾丁質?重組蛋白活性分析與酵素動力學 ............ 14
2.4.8 幾丁質?催化性部位結構模擬 ........................ 14
2.4.9 序列及演化樹分析 ........................... 14
第三章 結果 ............................... 16
3.1 質譜分析鑑定圍食膜蛋白 .................... 16
3.2 幾丁質結合蛋白的分類 ......................... 16
3.2.1 表皮蛋白 .............................. 16
3.2.2 圍食膜蛋白 ................................ 18
3.2.3 幾丁質代謝之酵素 .................... 18
3.3 鑑定小菜蛾幾丁質? ............................ 18
3.3.1 幾丁質?的分類 .......................... 19
3.3.2 幾丁質?序列分析及表現情形 ........................ 20
3.3.3 幾丁質?蛋白表現與生化活性 ................... 20
第四章 討論 ..................................... 23
4.1 質譜鑑定小菜蛾圍食膜蛋白.......................23
4.2 基因體搜尋小菜蛾圍食膜蛋白.....................24
4.3 基因體搜尋小菜蛾表皮蛋白.......................25

4.4 小菜蛾圍食膜與表皮蛋白之演化情形...............26
4.5 基因體搜尋小菜蛾幾丁質結合相關酵素..............27
4.6 其他小菜蛾幾丁質相關酵素.....................27
4.7 小菜蛾幾丁質?之序列分析與特性.................29
4.8 小菜蛾幾丁質?蛋白之生化特性..................32
第五章 結論與展望 ........................... 35
參考文獻 ...................................... 37
附錄
Supplemental Fig. 1. Phylogenetic analysis of the chitin binding domain of CPAP1s and
of the first CBD of CPAP3s and PMPs from Plutella xylostella.. ............................. 85
Supplemental Fig. 2. Complete genomic DNA sequence and cDNA deduced amino acid
sequence of PxCht8-2... ............................................................................................. 86
Supplemental table 1. The accession number for the CBPs used in phylogenesis. .............. 87
Supplemental table 2. Primer sets and conditions used for the RT-PCR in this Figure 4, 6
and 11....................................... 88
Supplemental table 3. The accession number for the chitinase proteins used in figure 14. . 89
Supplemental table 4. The accession number for the chitinase proteins used in figure 15 and
16. ....................................... 90
Supplemental table 5. Primer sets and conditions used for the RT-PCR in figure 19. ......... 91
Supplemental table 6. Primers and coditions used for the amplification of cDNA
corresponding to PxCht5, 7, 25-1 and 25-2. .................. 92
Supplemental table 7. Primers used to amplify the coding region fragments of PxCht5, 7,
25-1 genes. ....................................... 93
圖目錄
Fig. 1. Proteomic strategies for the identification of PM proteins from Plutela xylostella. . 50
Fig. 2. Typical 2-DE profiles of PM-Intergral and gut proteins.. ......................................... 51
Fig. 3. Phylogenetic analysis of the chitin binding domain of CPAP1s.. ............................. 52
Fig. 4. Expression of P. xylostella CPAP1 genes in different developmental stages and
different tissues as evaluated by RT-PCR.. ................................................................ 53
Fig. 5. Phylogenetic analysis of the full protein sequences of CPAP3s.. ............................. 54
Fig. 6. Phylogenetic analysis of the three chitin binding domains of CPAP3s. ................... 55
Fig. 7. Expression of P. xylostella CPAP3 genes in different developmental stages and
different tissues as evaluated by RT-PCR.. ................................................................ 56
Fig. 8. Phylogenetic analysis of the chitin binding domains of PMPs from P. xylostella and
M. sexta.. .................................................................................................................... 57
Fig. 9. Phylogenetic analysis of the chitin binding domains of PMPs from P. xylostella and T.
castaneum.. ................................................................................................................ 58
Fig. 10. Phylogenetic analysis of the chitin binding domains of PMPs from P. xylostella, M.
sexta and from T. castaneum.. ................................................................................... 59
Fig. 11. Expression of P. xylostella PMP genes in different developmental stages and
different tissues as evaluated by RT-PCR.. ................................................................ 60
Fig. 12. Phylogenetic analysis of the chitin binding domains of chitinases (Chts) from three
different insect species.. ............................................................................................. 61
Fig. 13. Phylogenetic analysis of the chitin binding domain of chitin deacetylases (CDAs)
from three different insect species.. ........................................................................... 62
Fig. 14. Phylogenetic analysis of RACE-PCR cloned chitinase proteins based on catalytic
domain sequences.. .................................................................................................... 63
Fig. 15. Phylogenetic analysis of lepidopteran chitinase proteins (NJ method) based on
catalytic domain sequences.. ..................................................................................... 64
Fig. 16. Phylogenetic analysis of lepidopteran chitinase proteins (ML method) based on
catalytic domain sequences. ...................................................................................... 65
Fig. 17. Domain architecture of the chitinase proteins of Plutella xylostella. ..................... 66
Fig. 18. Conserved regions in catalytic domains of chitinase proteins in Plutella xylostella..
................................................................................................................................... 67
Fig. 19. Expression of P. xylostella chitinase genes in different developmental stages and
different tissues as evaluated by RT-PCR. ................................................................. 68
Fig. 20. SDS-PAGE and Western blot analysis of three recombinant chitinases from E. coli..
................................................................................................................................... 69
Fig. 21. SDS-PAGE and Western blot analysis of two recombinant chitinase from yeast.. . 70
Fig. 22. SDS-PAGE and Western blot analysis of two recombinant chitinase expressed in
insect cell.. ................................................................................................................. 71
Fig. 23. Effects of pH and temperature on the purified recombinant PxCht5 and PxCht25-1
activities using the yeast system.. .............................................................................. 72
Fig. 24. Effects of pH and temperature on the purified recombinant PxCht7 and PxCht25-1
activities using the baculovirus-insect system.. ......................................................... 73
Fig. 25. Structure prediction of PxCht5 catalytic domain by using SWISS-MODEL. ........ 74
Fig. 26. Structure prediction of PxCht25-1 catalytic domain by using SWISS-MODEL. ... 75
Fig. 27. Homology structures of PxCht5 and PxCht25-1.. ................................................... 76
x
表目錄
Table 1. Identification of PM proteins by ESI-Q-TOF. ........................................................ 77
Table 2. Plutella xylostella genes encoding proteins with ChtBD2 domains.. ..................... 78
Table 3. Consensus of conserved cysteines (C) and spacings (X) of the ChtBD2 chitin
binding domain (CBD) for each category of chitin binding protein (CBP).. ............ 79
Table 4. Information on genes encoding chitinase and chitinase-like proteins searched from
P. xylostella genome and transcriptomic databases. .................................................. 80
Table 5. The estimated molecular mass of purified proteins. ............................................... 81
Table 6. Kinetic parameters of two proteins expressed in yeast system and
baculovirus-insect system. ......................................................................................... 82
Table 7. Information on genes encoding other chitin-associated enzyme searched from P.
xylostella genome databases. ..................................................................................... 83
Table 8. The predicted post-translational modification on the P. xylostella chitinases. ....... 84
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指導教授 黃榮南 高永旭(Rong-Nan Huang Yung-Hsi Kao) 審核日期 2017-1-24
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