Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/30983
標題: 鏈黴菌Streptomyces griseobrunneus S3幾丁質分解酵素基因之分子選殖及抗病性轉基因蕃茄與菸草之製作
Molecular cloning of chitinase genes from Streptomyces griseobrunneus S3 (SGS3) and the preparation of transgenic tomato and tobacco with improved disease resistance
作者: 楊尚書
Shang-Shu, Yang
關鍵字: Streptomyces griseobrunneus;Streptomyces griseobrunneus;ChiF chitinase;chitinase;transgenic plant;fluorescent microscopy;ChiF幾丁質分解酵素;轉基因植物;幾丁質分解酵素;螢光顯微鏡
出版社: 植物病理學系
摘要: 
本論文由已證實於植物病害防治上,深具應用潛力之Streptomyces griseobrunneus S3菌株(SGS3),成功選殖ChiA、ChiC與ChiD三個family 18幾丁質分解酵素部分基因序列,與一個family 19幾丁質分解酵素基因ChiF之部分核酸序列。試驗中初步檢視膠狀幾丁質添加對SGS3菌株內幾丁質分解酵素表現之影響,結果顯示ChiA、ChiC、ChiF均有短暫表現的現象,而隸屬於family 19之ChiF基因,則於所有測試SGS3幾丁質分解酵素中表現最強。本試驗中調查family 19幾丁質分解酵素,於台灣本土性具幾丁質分解能力鏈黴菌菌株之存在情形,以聚合酵素連鎖反應法(PCR),配合family 19幾丁質分解酵素專一性引子對的應用檢測,證實於21個供試菌株中,有11個可檢測到此酵素對應基因之存在,而無法測得之菌株,進一步以南方漬染檢測法,亦無此酵素基因之反應條帶。由已知同類酵素之胺基酸序列分析其親緣關係,所獲得樹狀演化圖顯示,SGS3 ChiF與已證實可能由植物class IV幾丁質分解酵素演化而來之S. griseus ChiC、S. coelicolor ChiF與Nocardiopsis prasina chiB等均具高度同源性。為闡明family 19幾丁質分解酵素於SGS3抗真菌特性之分子機制,試驗中選殖ChiF之全長核酸序列,經解序後證實共計含891 bp,所轉譯蛋白含296個胺基酸,分子大小約為31.5 kDa。此基因5’端之幾丁質結合區(ChtBD),具有與一般細菌幾丁質分解酵素type 3 ChtBD同樣之保留序列AKWWTQ。基因上游啟動子附近,並含有一已被證實與基因表現之幾丁質誘導與葡萄醣抑制調控攸關之核酸鹽基重複序列(T/A)GGTC(T/C)AGACC(T/A)。本研究已由SGS3 ChiF轉基因大腸桿菌中,成功萃取此ChiF重組蛋白,利用FPLC純化之蛋白製備,並進而製作成功其免疫檢測用多元抗血清。另利用高效率液相層析法(HPLC),檢測經ChiF蛋白裂解之幾丁質基質,證實ChiF為一內切幾丁質分解酵素,其可剪切4-methylumbellifery (4-Mu) triacetylchitotrioside成為一個雙醣和一個帶有色基的單醣,而無法剪切4-Mu diacetylchitobioside,而酵素最佳作用酸鹼值為pH 6.0。以試驗中所獲得之ChiF專一性抗血清,嘗試瞭解屬於family 19的幾丁質分解酵素ChiF SGS3對Rhizoctonia solani AG4與Pythium aphanidermatum兩主要土傳性病原真菌的超寄生作用中所扮演之角色,利用SGS3-ChiF專一性抗血清以免疫螢光標示法檢測發現,沿SGS3盤據部位均可見顯示ChiF存在之螢光反應,其中尤以在細胞壁含幾丁質的R. solani AG4菌體上可測得的螢光反應較強,而在細胞壁不含幾丁質的P. aphanidermatum菌絲體上,其螢光反應即相對較弱,本報告為首度在超寄生作用部位以免疫螢光檢測證實屬family 19的幾丁質分解酵素於鏈黴菌超寄生作用之關連性。另以所選殖之ChiF基因製作轉型蕃茄與菸草,以期能提升作物對於病害之抵抗性。試驗中以改造過之pCAMBIA 1301為轉型載體,並配合農桿菌之應用,進行轉基因作物之製備。試驗中成功選殖12株轉型蕃茄株系,經以北方雜合反應及西方轉漬反應與幾丁質活性染色,證實ChiF基因於蕃茄植株體內表現正常,於誘導抗病機制探討方面,轉型之蕃茄經處理1%膠狀幾丁質及幾丁質分解殘基,三天後即有PR-1基因表現,顯示轉型蕃茄以幾丁質處理後能促進抗病基因之表現。於菸草轉型方面,將源自於阿拉伯芥xyloglucan transferase基因外泌訊號核酸序列,以基因工程方式銜接於ChiF基因前端,使標的蛋白能以外泌狀態作用於試圖入侵之病原菌,利用相同轉型步驟,共計有19轉型株系株成功分化成為植株,經以北方轉漬法檢測,僅有11個轉型株系ChiF基因表現正常,試驗中選取1號及8號轉型分離株自交二代苗進行後續試驗,共計約有54% (line 1)至64% (line 8)二代苗ChiF基因表現正常,經澆灌處理膠狀幾丁質後,轉基因菸草於第10~14天具有PR-1基因之表現,顯示ChiF蛋白有助於分解膠狀幾丁質,其分解殘基進而可誘導植株產生抗病反應。而於抗病性測試方面,經接種白星病菌後,發現轉型菸草能有效降低壞疽病斑的形成及減緩病勢發展。由兩種轉型作物之抗病基因誘導表現及於真菌性病害防治應用性初步檢測結果,顯示此基因未來於作物病害綜合防治上深具應用價值。

The molecular characteristics of Streptomyces chitinases with apparent function in antifungal activity of Streptomyces griseobrunneus S3 (SGS3) were investigated. Partial sequence of family 18 chitinase genes ChiA, ChiC, ChiD and a family 19 chitinase gene ChiF were each respectively amplified and cloned. A screening on the expression of the gene with colloidal chitin application indicated SGS3 ChiF was preferentially expressed. A survey on the distribution of family 19 chitinase gene among Taiwan native chitinolytic Streptomyces strains was conducted. Among 21 tested strains, 11 were detected positive by polymerase chain reaction with the use of a family 19 chitinase gene specific primer pair. A further attempt to detect ChiF from these negative strains by southern analysis using ChiF specific probe was also negative. A phylogenetic analysis revealed that the cloned gene was highly homologous to the comparative gene known on S. griseus, S. coelicolor, and Nocardiopsis prasina, which might be adapted evolutionarily from the class IV chitinase of plant system. In order to elucidate the molecular basis of SGS3 family 19 chitinase in antifungal activity, the full-length of ChiF gene was cloned. Sequence analysis revealed that ChiF gene contains 891 bp which encodes a 296 amino acid peptide with a molecular weight around 31.5 KDa. The gene contains a chitin binding domain (ChtBD) with ChtBD consensus sequence AKWWTQ. On its upstream was a promoter region with characteristic direct repeat sequence (T/A)GGTC(T/C)AGACC(T/A) known to be critical in chitin induction and glucose repression. From a SGS3 ChiF transformed Escherichia coli RosettaTM (DE3), the ChiF recombinant protein with a predicted size around 31.5 KDa was extracted and purified by Fast Performance Liquid Chromatography. The polyclonal antibody for immuno-detection of the recombinant protein was successfully prepared. Product analysis by HPLC indicated that the SGS3 chiF protein is an endochitinase which converts 4-methylumbellifery (4-MU) triacetylchitotrioside to diacetylchitobioside and a nonfluorescent 4-MU derived acetlyglucosaminide, whereas fails to digest 4-MU diacetylchitobioside. The enzyme showed maximum activity at pH around 6.0. With the antiserum we got, we also explored the role of ChiF in mycoparasitism using Rhizoctonia solani AG4 and Pythium aphanidermatum as targeted fungi. The involvement of ChiF in the observed mycoparasitism was well illustrated by an immuno-fluorescent microscopy where that SGS3-ChiF specific polyclonal antibody was applied. The fluorescent signal indicating the expression of ChiF gene was detected along the contact interface of SGS3 on the host mycelium. The detected signal was strong especially among those colonizing on R. solani; the fluorescence detected from that on P. aphanidermatum was comparatively low. This is a first report of in situ detection of family 19 chitinase gene expression illustrating the importance of ChiF as a factor contributing to the mycoparasitism. The preparation of transgenic tomato and tobacco was attempted using ChiF gene cloned. By use of pCAMBIA 1301 as a transformation vector, the preparation of ChiF transgenic lines was successful via the Agrobacterium-mediated transformation. Twelve transgenic tomato lines carrying the target gene were successfully developed. The expression of the gene in these transgenic lines was demonstrated by both northern and western blotting analyses. In a mini-culture system established in a growth chamber, the application of ChiF digested colloidal chitin led to significantly enhanced elicitation, among the transgenic lines, of de novo expression of PR-1 gene about 3 days after treatment. For tobacco transformation, a secretary signal sequence of xyloglucan transferase gene adapted from Arabidopsis was added to forefront the target gene aiming to enforce the antifungal characteristic of the enzyme upon the host parasite confrontation. By the same strategy, a total of 19 transgenic lines were developed; and among them 11 were detected positive in ChiF gene expression in a followed northern analysis. The seeds collected from self-cross derived progenies of line 1 and line 8 germinated normally; and approximately 54% (line 1) to 64% (line 8) of the seedlings grown in the greenhouse showed ChiF gene expression in the foliar tissues. Upon drenching application of colloidal chitin, all the R1 progenies of both line 1 and line 8 transgenic tobaccos showed greatly enhanced expression of PR-1 gene in the foliar tissue about 10-14 days after treatment-indicating the enhanced resistance gene expression was functioning. By challenge inoculation with Cercospora nicotianae (causal agent of tobacco frog-eye disease), it was further shown that the necrotic symptom development on both transgenic lines was greatly retarded and alleviated comparing to that on the nontransgenic control plants. The accumulated evidence indicated clearly the targeted ChiF gene as a valuable tool for combating the diseases caused by wide spectrum of plant pathogenic fungi.
URI: http://hdl.handle.net/11455/30983
Appears in Collections:植物病理學系

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