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標題: 鏈黴菌Streptomyces griseobrunneus S3菌株所產生ChiA幾丁質分解酵素分子特性及與其抗真菌性之關係
Molecular characteristics of ChiA chitinase of Streptomyces griseobrunneus S3 and the role of ChiA in antifungal activity
作者: 黃琇屏
Huang, Shiou-Ping
關鍵字: 鏈黴菌
Streptomyces
幾丁質分解酵素
chitinase
出版社: 植物病理學系所
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Chitinase gene expression during mycoparasitic interaction of Trichoderma harzianum with its host. Fungal Genet. Biol. 26: 131-140.
摘要: 幾丁質分解性鏈黴菌Streptomyces spp.為一世界性分佈並廣為被發掘應用於植物病害生物防治上之有益微生物資源,Streptomyces griseobrunneus S3 (SGS3)菌株發展應用為典型例子之一,其已被證實可作為生物製劑並有效防治多種土壤傳播性真菌病害,而其病害防治效果則已知與其高度多樣化幾丁質及葡聚醣水解酵素之產生能力有關。楊氏於2005年已成功由SGS3菌株選殖一family 19幾丁質分解酵素基因chiF,並證實其於SGS3之抗真菌作用中攸關重要。本研究延續此一工作,進而由SGS3選殖其chiA基因並解析其生化特性及其真菌拮抗性中之重要特性。由研究結果證實SGS3 chiA全長計含有1668 bp,可轉譯出約555個胺基酸,對應蛋白分子大小約為61.2 kDa。利用NCBI blast軟體進行序列比對分析,得知其序列主要含一於N端之碳水化合物結合區間 (carbohydrate binding domain, CBM),及一位於C端之幾丁質催化活性區間(chitin catalytic domain);且分別與已知的S. coelicolor A3(2) chiA (GenBank accession no. BAB86375)及S. griseus chiI (GenBank accession np. BAB86375)具92 %及99%之相同性,由所轉譯酵素蛋白結構證實其與Saito氏等報導之ChiA同屬family 18 subfamily B幾丁質分解酵素。由序列分析發現SGS3 chiA含一group B之啟動子,且其附近含有已被證實與幾丁質誘導以及葡萄醣抑制等基因表現調控相關重複序列,然位於啟動子-35區域附近之核酸重複序列較之截至目前所知鏈黴菌之啟動子顯然有別。本研究將SGS3 chiA全長序列轉殖入大腸桿菌pET26b(+)表現載體,並利用此轉型菌株在大量誘導表現下成功萃取ChiA重組蛋白,繼之利用Superdex 200 HR 10/30分子篩管柱搭配ÄKTA FPLC系統純化此重組蛋白。經由螢光基質檢測法、電泳酵素圖譜分析、黏度計法、還原醣分析檢測及高效能液相層析(HPLC)法檢測反應產物,所獲結果證實SGS3 ChiA為兼具外切酵素(exo-chitinase)活性之內切幾丁質分解酵素(endo-chitinase)。檢測結果顯示利用由ChiF轉型大腸桿菌大量表現、萃取與純化之SGS3 ChiF重組蛋白同法進行酵素活性分析比較,ChiA可剪切4-methylumbellifery diacetylchitobiose (4-MU-GlcNAc2)成為一個雙醣及螢光色基;亦可剪切4-MU-triacetylchitotriose (4-MU-GlcNAc3)成為一個雙醣、一個單醣及螢光色基;此外以GlcNAc6為基質時,其早期產物以GlcNAc2與GlcNAc4為主,最終產物則主要為GlcNAc2。此幾丁質分解特性異於SGS3 ChiF;同樣為兼具內切及外切特性之幾丁質分解酵素,ChiF可剪切GlcNAc6成GlcNAc2、GlcNAc4與GlcNAc3,最終產物則以GlcNAc2為主。至於ChiA及ChiF對可溶及不可溶幾丁質分解特性則無明顯差異,由本研究中所獲試驗結果顯示此兩酵素均對可溶性幾丁質GlcNAc3分解力佳,而對glycol-chitin之分解力較弱,而ChiA與ChiF混合添加下經檢測其對不同幾丁質基質之作用則發現彼此間應無明顯協力作用。此外兩種酵素間特性上相異部份包括SGS3 ChiA最適反應pH值為5.0,最適反應溫度則為50℃,及酵素活性於添加鈣離子及鎂離子時可提高會受到促進;而SGS3 ChiF則最適反應pH值為5.0,最適反應溫度為40℃,且上述兩種離子的添加對其活性並無促進作用。為釐清此兩酵素於SGS3拮抗病原真菌之作用,本研究嘗試利用僅具ChiA catalytic domain (ChiAΔCBM)重組蛋白製作大白兔免疫專一性多元抗體,以分別標幟FITC與rhodamine之ChiAΔCBM與ChiF抗血清配合免疫螢光標示法檢測發現,纏據於兩病原真菌上之SGS3孢子均可見顯示ChiA與ChiF之螢光反應,值得注意的是於SGS3纏據於R. solani AG4上之菌絲仍可偵測到強烈之ChiF表現,卻無法偵測到ChiA存在之訊號,此結果顯示ChiA在SGS3之抗真菌性作用中所扮演之角色可能未若ChiF之重要。
The chitinolytic Streptomyces spp. are worldwide distributed beneficial microbial resources being actively explored as bio-agent for controlling plant diseases. The application of Streptomyces griseobrunneus strain S3 (SGS3) is one of the typical examples, its application as biofungicide for the control of various soil borne fungal diseases has been shown to be successful. And its effectiveness of disease control was found to due at least in part to the production of bountiful chitin and glucan hydrolytic enzymes. A family 19 chitinase gene chiF was cloned and shown to be one of the primed attributes contributing to the antifungal activity of SGS3 (Yang, 2005). In the present investigation, a full length chiA gene was cloned from SGS3 and its biochemical characteristics and biological significances especially the possible involvement in antagonistic activity of the test bacterium were explored. The cloned gene appeared to be 1668 bps in length which encoded a 555-amino acid composed protein molecule with molecular weight around 61.7 kDa. The sequence analysis by blast software of NCBI indicated that the cloned SGS3 chiA mainly consisted a carbonhydrate binding domain (CBM) at the N-terminal and a chitin catalytic domain on the C-terminal. The pairwise sequence comparison indicated that it shared a 84 % and 92 % identity to chiA of S. coelicolor A3(2) (GenBank accession no. BAB86375) and chiI of S. griseus (GenBank accession no. BAA75642) respectively. The encoded enzyme thus appeared to belong to chi18bA (subfamily B of family 18 chitinase A) as that suggested by Saito (2000). A promoter region belonging to group B was identified, in adjacent to the promoter region were some repetitive regulatory sequences relating to chitin induction and glucose repression. The repetitive sequences found close to the -35 upstream of promoter sequence appeared to be discrete as compared to Streptomyces group B promoters known to date. The full length of chiA has been transformed to Escherichia coli expression vector pET26b(+), and the recombinant ChiA protein was produced by over-expression of the transformant bacteria and purified by an ÄKTA FPLC system using with Superdex 200 HR 10/30 column. The serial analysis on the hydrolytic activity including that by application of specific fluorochrome-linked oligo-chitin substrates, by electrophoresis zymogram investigation, by viscometry, by reducing sugar assay and by high performance liquid chromatography (HPLC) of the reaction products, indicated clearly the studied SGS3 ChiA was endo-chitinase with certain exo-enzyme activity. The analysis was performed with the use of recombinant SGS3 ChiF purified from a SGS3 chiF transformant E. coli as a comparison. From the data herein presented, the enzyme appeared to cut a 4-methylumbellifery linked chitobiose (4-MU-GlcNAc2) into a chitobiose (GlcNAc2) and a free fluorochrome, and a 4-MU-GlcNAc3 into a GlcNAc2, a chitose monomer GlcNAc and the free fluorochrome. With the provision of GlcNAc6, the primary early product appeared to be GlcNAc2 and GlcNAc4, whereas the final product was primarily the disaccharide GlcNAc2. The hydrolytic activity of SGS3 ChiA appeared to differ greatly to that of SGS3 ChiF. The later was known to have endo- and exo-chitinase activity, and with the provision of GlcNAc6, the data herein presented indicated that the primary early product was GlcNAc2, GlcNAc3 and GlcNAc4, whereas the final product was primarily GlcNAc2.and GlcNAc. The activity of SGS3 ChiA was shown to have an optimum pH at 5.0, optimum temperature at 50 ℃, and was promoted by the presence of dications Mg2+ and Ca2+; the compared SGS3 ChiF was shown to have optimum pH at 5.0, an optimum temperature at 40 ℃, and the activity was not affected by the above shown dications. Also, both enzymes were shown to work best on artificial soluble chitin, fair on colloidal chitin and chitosan, and very weak on glyco-chitin. To illustrate the role of both enzymes in the antagonistic activity against fungal pathogen, a chitin binding domain truncated SGS3 ChiA designated as chiAΔCBM was constructed and cloned to E. coli for preparation of ChiAΔCBM recombinant protein. And by immunization of rabbit, the ChiA specific polyclonal antibody was prepared. The in situ detection of chitinase expression during the mycoparasitism of SGS3 was performed by differential staining fluorescent microscopy with the use of FITC labeled ChiAΔCBM specific IgG and rhodamine labeled ChiF. The results obtained revealed that upon the infection of SGS3 on Rhizoctonia solani AG4, the colonization of the spore biomass on the fungal thallus was consistently accompanied with the expression of both ChiF and ChiA. It was worth noting however that the expression signal of ChiF detected from the mycelial extension of SGS3 was still quite strong, but that of ChiA appeared to be trivial. The results seemed to downplay the role of ChiA in the antagonistic effectiveness of the target bacterium.
URI: http://hdl.handle.net/11455/31252
其他識別: U0005-2208200813535900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-2208200813535900
顯示於類別:植物病理學系

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