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研究生:方俊偉
研究生(外文):Chun-wei Fang
論文名稱:利用PCR與RT-PCR技術偵測瓜類細菌性果斑病菌之研究
論文名稱(外文):Studies on detection of Acidovorax avenae subsp. citrulli by using PCR and RT-PCR techniques
指導教授:王惠亮
指導教授(外文):Hui-Liang Wang
學位類別:碩士
校院名稱:國立高雄師範大學
系所名稱:生物科學研究所
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:2001
畢業學年度:90
語文別:中文
論文頁數:84
中文關鍵詞:細菌性果斑病瓜類細菌性果斑病細菌性果斑病菌瓜類細菌性果斑病菌細菌性果斑果斑病果斑病菌
外文關鍵詞:AcidovoraxAcidovorax avenaeavenaecitrulliAcidovorax avenae subsp. citrulli
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本研究利用網路上基因資料庫中與瓜類細菌性果斑病菌Acidovorax avenae subsp. citrulli相關的基因序列資料,設計出3條10 mer引子,之後以RT-PCR反應技術選殖瓜類細菌性果斑病菌Aa31之cDNA。在使用引子對Aap5/Aap6的RT-PCR產物中,以約1600 bp的cDNA片段最明顯。在使用引子對Aap5/Aap61的RT-PCR產物中,以約900 bp的cDNA片段最明顯。將兩cDNA片段回收,用20 mer加長引子對以PCR增幅此兩段cDNA片段,其中以約900 bp的cDNA片段增幅之結果較好。因此選擇約900 bp的cDNA片段做核酸譯讀定序,結果顯示在扣除掉外加部份的長度後,從Aa31選殖到的cDNA片段長度為887 bp。在分析此段887 bp cDNA片段之核酸序列後,從不同的開放解讀架構 ( open reading frame ) 中找出六段完整的胺基酸序列Ama01、Ama02、Ama03、Ama04、Ama05、Ama06,其中Ama01、Ama02、Ama03、Ama05、Ama06之胺基酸序列與網路上基因資料庫內已註冊之胺基酸序列皆無明顯的相似,只有Ama04之胺基酸序列與許多細菌與病毒內的轉錄調控因子 ( transcriptional regulator ) 、轉錄抑制因子 ( transcriptional repressor ) 和細胞分裂控制蛋白 ( cell division control protein ) 之胺基酸序列有一定程度的相似,由此可知Ama04可能在瓜類細菌性果斑病菌Aa31之生理上扮演著重要的調控角色。本研究亦從此段887 bp cDNA片段之核酸序列中設計出一對偵測用之引子對L1/R1,用引子對L1/R1以PCR反應偵測含Aa31在內的十四個細菌性果斑病菌菌株,結果除了Aa31必然可以增幅出360 bp DNA片段之外,Aa3, Aa27, Aa29, Aa30, Aa32, Aa33也可以增幅出360 bp DNA片段,其他本研究中所使用的果斑病菌菌株 ( 包括所有從西瓜分離到的菌株 ) 則不能反應。而非果斑病菌之其他八種植物病菌用引子對L1/R1以PCR反應偵測,結果亦皆無法增幅出360 bp DNA片段。用引子對L1/R1以PCR反應偵測Aa31之全量DNA時,靈敏度可達到100 pg,而以PCR反應偵測Aa31之菌數時,靈敏度可達到103 cfu,若以RT-PCR反應偵測Aa31之菌數時,靈敏度亦可達到102 cfu。

Three different primers [ one upstream primer ( Aap5 ) and two downstream primers ( Aap6 and Aap61 ) ] selected from Acidovorax avenae-associated nucleotide sequences in Genbank database of National Center for Biotechnology Information ( NCBI ) on internet were used to clone specific cDNA fragments of A. avenae subsp. citrulli Aa31 by using RT-PCR. cDNA products of ~1600 bp with Aap5/Aap6 primers and ~900 bp with Aap5/Aap61 primers were obtained by using RT-PCR. For further nucleic acid sequence analysis, the two cDNA fragments were amplified with PCR using elongated primers. The ~900 bp cDNA fragment was used for sequencing of nucleic acid sequence. This 887 bp nucleotides sequence of cDNA fragment were analyzed. Six open reading frames were found, designed as Ama01, Ama02, Ama03, Ama04, Ama05, and Ama06, respectively. Among them, Ama04 had 40% identities and 61% positives with transcriptional regulators, transcriptional repressors, and cell division control proteins of some bacteria and phages by searching with NCBI Genbank database. The others had no significant identities. Therefore, Ama04 might play an important physiological role in Aa31. A primer pair L1/R1 was also designed from the nucleic acid sequence of 887 bp cDNA fragment for detection of Aa31. 14 strains of A. avenae subsp. citrulli and 8 kinds of other plant pathogenic bacteria were tested by PCR amplification with primer pair L1/R1. The result indicated that a distinct band of 360 bp was amplified with primer pair L1/R1 from Aa31, a less obvious band of 360 bp was amplified with primer pair L1/R1 from Aa3, Aa27, Aa29, Aa30, Aa32, Aa33 of A. avenae subsp. citrulli, and no amplification from watermelon strains Aa9, Aa24, Aa60 of A. avenae subsp. citrulli and other plant pathogenic bacteria. The minimum amount of DNA from A. avenae subsp. citrulli Aa31 that could be amplified by PCR was 100 pg. Sensitivity of PCR and RT-PCR for detection of cells of A. avenae subsp. citrulli Aa31 with primer pair L1/R1 were 103 cfu and 102 cfu, respectively. From the result, PCR and RT-PCR were able to detect A. avenae subsp. citrull effectively. Genes of the 887 bp cDNA fragment could be potentially used for the further research of A. avenae subsp. citrulli.

中文摘要…………………………………………………………I
英文摘要…………………………………………………………III
壹、前言…………………………………………………………1
貳、材料與方法…………………………………………………10
一、菌株來源……………………………………………………10
二、細菌全量RNA的萃取、濃度測定與膠體電泳分析…………10
(一)、萃取細菌全量RNA……………………………………10
1、菌株培養…………………………………………10
2、萃取全量RNA……………………………………13
(二)、RNA濃度及純度測定…………………………………13
(三)、RNA之膠體電泳分析…………………………………14
三、瓜類細菌性果斑病菌菌株Aa31進行RT-PCR與cDNA片段之定序分析…14
(一)、RT-PCR ( 反轉錄聚合酵素連鎖反應 ) ………………14
1、10 mer引子對的設計…………………………………14
2、進行RT-PCR反應………………………………………15
(二)、RT-PCR產物之膠體電泳分析……………………………17
(三)、cDNA片段之回收…………………………………………17
1、使用Quick-Pik Electroelution Capsule
( Stratagene ) 回收cDNA…………………………17
2、使用Low Gelling Temperature Agarose
Gel Method ( 低凝結點的洋菜水平膠體
) 回收cDNA…………………………………………18
(四)、回收之cDNA使用加長之引子以PCR增幅…………………20
1、加長引子之設計………………………………………20
2、以PCR增幅cDNA片段…………………………………22
3、PCR產物之膠體電泳分析……………………………23
(五)、瓜類細菌性果斑病菌菌株Aa31 cDNA片段
核酸序列之譯讀…………………………………………23
四、瓜類細菌性果斑病菌菌株Aa31 cDNA片段核酸序列之應用…23
(一)、從譯讀出的瓜類細菌性果斑病菌菌株Aa31
cDNA片段之核酸序列設計引子對…………………24
(二)、瓜類細菌性果斑病菌菌株Aa31之三
種全量DNA萃取方法之比較分析……………………24
1、萃取病菌全量DNA之三種方法…………………24
2、以PCR測定引子對L1/R1之適當黏合溫度………28
3、三種萃取方法所得之全量DNA之PCR結果比較分析…29
(三)、以PCR測定引子對L1/R1之專一性與靈敏度………30
1、以PCR測定引子對L1/R1之專一性………………30
2、以PCR測定引子對L1/R1對瓜類細
菌性果斑病菌之靈敏度…………………………30
(四)、以RT-PCR測定引子對L1/R1對瓜類細菌性
果斑病菌之靈敏度……………………………………32
1、萃取全量RNA……………………………………32
2、RT-PCR……………………………………………33
(五)、PCR與RT-PCR產物之膠體電泳分析…………………34
參、結果…………………………………………………………35
一、細菌全量RNA的抽取…………………………………………35
二、瓜類細菌性果斑病菌菌株Aa31進行RT-PCR與cDNA片段之定序分析…35
(一)、RT-PCR產物之膠體電泳分析………………………35
(二)、瓜類細菌性果斑病菌菌株Aa31 cDNA片段之PCR增幅、選擇與回
收……………………………………………………37
(三)、核酸序列之譯讀定序與分析………………………41
三、瓜類細菌性果斑病菌菌株Aa31 cDNA片段核酸序列之應用…53
(一)、測定引子對L1/R1對瓜類細菌性果斑病
菌的適當黏合溫度之PCR試驗結果…………………53
(二)、瓜類細菌性果斑病菌菌株Aa31之三種
全量DNA抽取方法之比較分析………………………53
(三)、測定引子對L1/R1對瓜類細菌性果斑病
菌的專一性之PCR試驗結果…………………………54
(四)、測定引子對L1/R1對瓜類細菌性果斑病
菌的靈敏度之PCR試驗結果…………………………55
1、以瓜類細菌性果斑病菌之DNA量做
測試………………………………………………55
2、以瓜類細菌性果斑病菌之病菌數做
測試………………………………………………61
(五)、測定引子對L1/R1對瓜類細菌性果斑病
菌的靈敏度之RT-PCR試驗…………………………61
肆、討論…………………………………………………………65
伍、參考文獻……………………………………………………75

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