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研究生:陳冠蓓
論文名稱:白色海芋軟腐病之病原鑑定、分子類型區分、及pectatelyase基因的表現及應用
論文名稱(外文):Identificaiton and molecular typing of white flowered calla lily soft rot pathogen, and pectate lyase gene expression and application
指導教授:李永安李永安引用關係
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生命科學系碩士班
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:140
中文關鍵詞:白色海芋軟腐病病原鑑定
外文關鍵詞:molecular typingsoft rot pathogenpectate lyase
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在陽明山竹子湖種植的白色海芋中,發現有軟腐病徵的植株,從得病組織中,分離出病原細菌,經接種實驗結果,確定所分離出的細菌可感染白色海芋,並造成軟腐病徵。生理生化測試結果,該病原細菌為Gram negative,兼性厭氧,對erythromycin敏感,不具有oxidase活性、具有lecithinase活性,及可產生indole,不能利用trehalose作為碳素源,鑑定結果為Erwinia chrysanthemi,進一步利用16S rDNA所設計的primer進行PCR,將PCR產物接在pGEM-T vector,送入E.coli DH5a中,再將其clone作序列定序,最後以此病原菌的16S rRNA序列(AY360397)和E. chrysanthemi之16S rRNA序列作比對,結果具有98﹪的相同度。另外藉由indC 和tryptophanase gene為探針,進行Southern hybridization,其探針可以和E. chrysanthemi產生雜交結果,不會和E. carotovora subsp. carotovora產生雜交結果;而利用pelZ基因所設計的 primer進行PCR,可以在E. chrysanthemi 擴增出1.2kb的DNA片段,但是在E. carotovora subsp. carotovora不能擴增出產物,因此可以區分E. chrysanthemi和E. carotovora subsp. carotovora,經由生理生化測試,16S rDNA序列分析,及特定基因之分子鑑定結果,確定感染白色海芋軟腐病的病原菌為E. chrysanthemi。將分離自不同寄主的E. chrysanthemi接種於白色海芋花梗,發現從白色海芋分離出的E. chrysanthemi致病力最強,從蝴蝶蘭分離出的E. chrysanthemi致病力次之,相對的從青蔥、芹菜分離出的E. chrysanthemi致病力最弱,而從白菜和馬鈴薯所分離出的E. carotovora subsp. carotovora致病力也最弱。將白色海芋分離菌株E. chrysanthemi S3-1接種於不同的植物寄主,結果發現,白色海芋分離菌株E. chrysanthemi S3-1會感染馬鈴薯、蕃薯、洋蔥、紅蘿蔔、蝴蝶蘭、青椒、芹菜、及彩色海芋這八種植株。將E. chrysanthemi S3-1和E. carotovora subs. carotovora CS3-2,分別培養在28℃和37℃的水中,發現不論是在28℃或37℃水中,白色海芋分離菌株E. chrysanthemi S3-1的存活時間,都較彩色海芋分離菌株CS3-2的存活時間久。為了了解白色海芋的軟腐病,是否為台灣其他植物寄主的軟腐病菌所引起,因此進一步利用Restriction Fragment Length Polymorphism (RFLP)、PCR-RFLP及 pulsed field gel electrophoresis(PFGE),對台灣不同植物所分離出的E. chrysanthemi進行分子類型(molecular typing)區分,在RFLP 的實驗中,藉由indC、tryptophanase、pel AED,和pel Z基因為探針,進行Southern hybridization,結果發現白色海芋和其他植物寄主的E. chrysanthemi為兩種不同的類型(pattern);在PCR-RFLP 的實驗中,利用pelZ gene序列所設計的 primer進行PCR,將PCR的產物分別以Ahd I和Pst I酵素作用,結果也發現白色海芋和其它植物寄主的E. chrysanthemi為兩種不同的類型(pattern);在pulsed-field gel electrophoresis的實驗中,將不同植物所分離出的E. chrysanthemi和E. carotovora subsp. carotovora,其total DNA以Xba I酵素作用之後,進行脈衝式電泳,結果發現不同植物寄主的E. chrysanthemi各有不同的類型(pattern),經由以上3種molecular typing方法的分析,皆可以區分出從白色海芋分離出的E. chrysanthemi和其它植物所分離出的E. chrysanthemi為不同的分子類型,而E. chrysanthemi和E. carotovora subsp. carotovora的分子類型差異更大。由於白色海芋先前未發現有軟腐病,經由致病力的檢測及分子類型的區分,可以推論出白色海芋的病原細菌,與原先國內已有的E. chrysanthemi有所不同,可能是國外新引入的菌種。
之前有文獻指出Erwinia spp.藉由產生pectate lyase破壞植物細胞壁,進而造成植物軟腐病,而被破壞的植物細胞壁,分解成小片段的oligosaccharides,這些oligosaccharides將會引起植物的抗病反應,產生一些抗菌或殺菌物質,但是當植物產生出這些物質時,Erwinia spp.的數量已經太多,無法被毀滅,因此如果能讓健康的植物細胞壁,在平時就分解一些少量的小片段的oligosaccharides,使植物平常即產生一些抗菌或殺菌物質,就可以達到抗軟腐病的效果。由於白色海芋沒有完整的組織培養和轉基因技術,而蝴蝶蘭有完整的組織培養和轉基因方法,並且蝴蝶蘭軟腐病的病原菌亦為E. chrysanthemi,因此計畫將E. chrysanthemi 的pectate lyase基因pelE及pelZ分別送入蝴蝶蘭中,以培育可以抗軟腐病的蝴蝶蘭品種。自E. chrysanthemi將pelE及pelZ選殖出來,接在pET29a vector中,送入E.coli BL21 (DE3),可產生47.13-kDa的PelE和48.38-kDa的PelZ酵素,再進一步純化出47.13-kDa的PelE和48.38-kDa 的PelZ酵素,偵測出其酵素具有活性,將具有活性的酵素接種於蝴蝶蘭的葉子,發現可以誘發蝴蝶蘭對E. chrysanthemi的抗病效果,因此將pelE gene及pelZ gene接在pBI121 vector 中,正進行基因轉殖中。
Soft rot symptoms on white flowered calla lily (Zantedeschia aethiopica) were found in some nurseries in the Yang Ming Shan area, Taipei, Taiwan. Isolations from diseased flower stems consistently yielded bacterial colonies. Koch's postulates were completed by injecting bacterial suspensions (108 CFU/ml) into stems of white flowered calla lily. Control plants were inoculated with sterile distilled water. Symptoms developed 1-2 days in all inoculated plants and appeared to be identical to those observed on diseased material in nurseries. All of the control plants did not become rotten. Ten representative isolates were chosen for further characterization. All isolates were gram-negative rods, facultatively anaerobic, sensitive to erythromycin (25 g/ml), negative for oxidase, and positive for tryptophanase (indole production), and lecithinase. The isolates cannot produce acid from trehalose. This result revealed that this pathogen was identified as Erwinia chrysanthemi. Primers designed from 16S rRNA gene sequence were further utilized to perform PCR. PCR product was cloned and sequenced. Sequence analysis of 16S rRNA gene (AY360397) of this pathogen, revealed 98% of homology to 16S rRNA gene of E. chrysanthemi. By using indC and tryptophanase genes as probes in Southern hybridization, it was found that E. chrysanthemi showed positive result, but not with E. carotovora subsp. carotovora. A PCR primer set (pelZ exp) was designed based on conserved regions of pelZ gene, and a 1.2 kb PCR product can be amplified from E. chrysanthemi, but not from E. carotovora subsp. carotovora. Therefore, indC, tryptophanase probes, and the pelZ exp primer set are E. chrysanthemi-specific and can be used to identify E. chrysanthemi. Base on the results of physiological and biochemical test, sequence analysis of 16S rDNA gene, and molecular detection of specific genes, confirmed that this pathogen which infected white flowered calla lily and caused soft rot disease is E. chrysanthemi and named this strain as S3-1. By inoculating pedicels of white flowered calla lily with E. chrysanthemi isolated from different hosts, it was found that E. chrysanthemi which was isolated from white flowered calla lily showed the most serious symptom, pathogenicity of E. chrysanthemi isolated from phalaenopsis is the second, and those isolated from green onions and celery, their pathogenicity is weaker. In the other hand, E. carotovora subsp. carotovora isolated from the Chinese cabbage and potato also showed weak pathogenicity after inoculated to the pedicels of white flowered calla lily. By inoculating E. chrysanthemi strain S3-1 to different plants, we found that this pathogen can cause soft rot disease in potato, sweet potato, onion, carrot, phalaenopsis, sweet pepper, and celery. E. chrysanthemi S3-1 and E. carotovora subsp. carotovora CS3-2 were cultured in 28℃and 37℃ water respectively. We found that E. chrysanthemi S3-1 survive longer than E. carotovora subsp. carotovora CS3-2 either in 28℃ or 37℃ water. In order to understand whether the soft rot disease of the white flowered calla lily was caused by E. chrysanthemi of other plant hosts in Taiwan, molecular typing methods including Restriction Fragment Length Polymorphism (RFLP), PCR-RFLP, and pulsed field gel electrophoresis (PFGE)were used. In the experiment of RFLP, indC, tryptophanase, pel AED, and pelZ genes were used as probes to perform Southern hybridization, it was found that E. chrysanthemi from white flowered calla lily and other plants host are two different patterns. A PCR primer set was designed based on the conserved regions of pelZ gene. PCR-RFLP study was undertaken by digesting the amplified fragment with Ahd I and Pst I enzymes. The digestion pattern revealed that E. chrysanthemi of white flowered calla lily is different from E. chrysanthemi of other hosts. In the PFGE experiment, the banding patterns obtained with enzyme Xba I digestion revealed significant differences among E. chrysanthemi and E. carotovora subsp. carotovora strains from different hosts. Results from the above-mentioned molecular typing methods, E. chrysanthemi isolated from the white flowered calla lily can be distinguished from those isolated from other plants as different molecule types. Besides, the molecule typing differences between E. chrysanthemi and E. carotovora subsp. carotovora are greater. Via pathogenesis test, and molecular typing, the E. chrysanthemi strain that caused soft rot diseases among white flowered calla lily is different from E. chrysanthemi strain found in Taiwan before, and due to the soft rot disease has not been found among the white flowered calla lily previously, so we suggested that the E. chrysanthemi strain which isolated from white flowered calla lily is newly introduced from abroad.
Soft rot Erwinia spp. causes the soft rot disease of plants by producing pectate lyase that able to degrade plant cell wall. The pectate lyase will lead to the realease of the oligosaccharides, these plant cell wall fragments function as elicitors to stimulate plant defense responses. We reasoned that the introduction of pectate lyase gene into plants would alter the situation to the benefit of the plant host by triggering pectate lyase synthesis, which is already at low bacterial cell densities. Due to lack of complete tissue cultivation and transgenic technology for white flowered calla lily (Zantedeschia aethiopica), while there are complete tissue culture and transgenic technique for phalaenopsis, and the soft rot disease pathogen of phalaenopsis is also E. chrysanthemi, therefore this study is to introduce pelE and pelZ of the pectate lyase gene of E. chrysanthemi into phalaenopsis separately, in order to cultivate the phalaenopsis which can resist the soft rot disease. The pelE and pelZ genes from E. chrysanthemi were cloned and the 47.13-kDa PelE enzyme and 48.38-kDa PelZ enzyme was produced in E. coli pET system separately. Although most of PelE and PelZ are insoluble and formed an inclusion body, a few soluble PelE and PelZ still existed in soluble portion and can be purified. The purified soluble PelE and PelZ have the pectate lyase activity. Phalaenopsis leaves were inoculated with these enzymes. We found that PelE and PelZ will induce phalaenopsis's defense responses to E. chrysanthemi. At this stage the pelE gene and pelZ genes have been cloned to pBI121 vector, and in the process of being transgened to phalaenopsis.
目 錄
第一章 白色海芋疑似軟腐病之病原鑑定
中文摘要--------------------------------
英文摘要---------------------------------------------------
前言------------------------------------------
材料與方法-------------------------------------------
菌種的取得、培養條件、儲存的方法--------------------------
自感病植物中分離菌株--------------------------------------------
軟腐病菌的鑑定--------------------------------------
1.軟腐病菌的接種實驗--------------------------------------
2.生理生化測試-------------------------------------------
3.16S rRNA基因選殖、定序及比對----------------------------
NJ(neighbor-joining method)劃親源關係的步驟-------------------
分子鑑定區分E .chrysanthemi和E. carotovora subsp. carotovora-------
1.以indC 和tryptophanase基因為探針進行南方雜合反應(RFLP)---
2.利用pelZ gene序列所設計的 primer進行PCR--------------------------
自台灣分離的軟腐病菌對白色海芋致病力強弱的檢測---------------------
白色海芋分離菌株S3-1之寄主範圍測試-------------------------------------
白色海芋分離菌株S3-1和彩色海芋分離菌株CS3-2,水中存活比較----
病原菌的分子類型(molecular typing)區分---------------------------------
1.specific gene-RFLP-------------------------------------------------
2. PCR-RFLP-------------------------------------------------
3. Pulsed-field gel electrophoresis------------------------------------
礎分生技術-------------------------------------------------------------
實驗結果---------------------------------------------------------------
自感染軟腐病徵的白色海芋中分離菌株---------------------------------------
白色海芋分離菌株對白色海芋的接種實驗-----------------------------------
白色海芋分離菌株S3-1生理生化測試結果-----------------------------------33
16S rDNA基因選殖、定序及比對結果------------------------------------------
分子鑑定區分E .chrysanthemi和E. carotovora subsp. carotovora結果------
1.軟腐病菌菌株以indC基因為探針進行南方雜合反應結果-----------------
2.軟腐病菌菌株以tryptophanase 基因為探針進行南方雜合反應結果---
3. 利用pelZ gene序列所設計的 primer進行PCR反應結果---------------
白色海芋軟腐病害的病原細菌鑑定結果--------------------------------------
自台灣分離的軟腐病菌對白色海芋致病力強弱的檢測結果------------------
白色海芋分離菌株S3-1之寄主範圍測試-------------------------------------
白色海芋分離菌株S3-1和彩色海芋分離菌株CS3-2水中存活比較結果----
. chrysanthemi之分子類型(molecular typing)區分結果------------------
Restriction Fragment Length Polymorphism (RFLP)------------------------
1.軟腐病菌菌株以indC基因為探針進行南方雜合反應結果-----------------
2.軟腐病菌菌株以tryptophanase 基因為探針進行南方雜合反應結果------
3.軟腐病菌菌株以pel AED基因為探針進行南方雜合反應結果------------
4. 軟腐病菌菌株以pel Z基因為探針進行南方雜合反應結果----------
PCR-RFLP---------------------------------------------------
1.pelZ 基因序列所設計的 primer進行PCR反應之產物以AhdI 酵
素作用結果-----------------------------------------------------------
2 pelZ 基因序列所設計的 primer進行PCR反應之產物以PstI酵
素作用結果----------------------------------------------------------------
pelZ基因分析比對結果-----------------------------------------------------
Pulsed-field gel electrophoresis---------------------------------------------------
1. 不同寄主來源之Erwinia chrysanthemi其pattern結果-----------------
2. 不同Erwinia spp.其pattern結果---------------------------------------------
白色海芋分離菌株S3-1鑑定結果---------------------------------------------
討論---------------------------------------------------
參考文獻------------------------------------------------------------
圖表------------------------------------------------------------------
第二章 pectate lyase基因的表現及應用
中文摘要----------------------------------------------------------
英文摘要---------------------------------------------------------------
前言----------------------------------------------
材料與方法-----------------------------------------------------------------
菌種的取得、培養條件、儲存的方法-------------------------------------------
序列分析------------------------------------------------------
將pelE和pelZ基因接入pET29a-----------------------------------------------
PelE和PelZ蛋白質之大量表現------------------------------------------------
PelE和PelZ蛋白質之純化---------------------------------------------------------
PelE和PelZ酵素活性測定------------------------------------------------------
PelE和PelZ酵素抗體活性測試---------------------------------------------------
1.Enzyme —linked immunosorbent assay (ELISA)-------------------------
2.Westrn blotting---------------------------------------------------------------
蝴蝶蘭的接種實驗-----------------------------------------------------
基礎分生技術-----------------------------------------------------------------
實驗結果------------------------------------------------------------------
自E. chrysanthemi選殖出pectate lyase 基因---------------------------------------
PelE和PelZ蛋白質大量表現-------------------------------------------------
PelE和PelZ蛋白質純化結果---------------------------------------------------
PelE和PelZ蛋白質活性測定----------------------------------------------------
PelE蛋白質抗體測試結果----------------------------------------------------
1.Enzyme —linked immunosorbent assay (ELISA)---------------------------
2.Westrn blotting-----------------------------------------------------------------
蝴蝶蘭葉子接種實驗結果------------------------------------------------
抗軟腐病之轉基因蝴蝶蘭-------------------------------------------------
討論----------------------------------------------------------------
參考文獻------------------------------------------------------------------
圖表----------------------------------

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