彩色海芋軟腐細菌Erwinia carotovora subsp. carotovora集約反應 N-acyl homoserine lactone synthase 基因之選殖及其轉殖菸草對Erwinia屬軟腐細菌之感受性

Abstract

集約反應 (quorum sensing)為細菌個體感應環境中自我族群大小之系統，細菌可藉此調控其基因之表現。自我誘導物 (autoinducer)為細菌集約反應之信號分子，在革蘭氏陰性細菌中之自我誘導物通常為N-acyl homoserine lactone (HSL)，HSL 可由HSL synthase生合成後釋放至菌體外以誘發集約反應。本研究進行彩色海芋細菌性軟腐病菌Erwinia carotovora subsp. carotovora (Ecc) ZL1 HSL synthase基因之選殖，並將其轉殖於菸草以探討HSL在軟腐細菌與寄主交互作用中所扮演之角色。依據Ecc SCC3193菌株之集約反應expI基因序列設計引子對expI-F/expI-R，並利用聚合酶鏈鎖反應 (polymerase chain reaction , PCR )對20個彩色海芋軟腐細菌Ecc菌株之DNA模板進行增幅，結果顯示所有菌株均可增幅出一約1.2 kb之DNA片段，進一步將ZL1菌株所增幅出之條帶回收及選殖後經解序並利用BLASTN比對，顯示所選殖之片段與資料庫Ecc 71菌株之hslI基因核酸序列有92%的相似度。將Ecc ZL1菌株之HSL synthase基因構築於pT7-7表現載體並轉型至Escherichia coli BL21 DE3中進行蛋白表現及電泳分析，結果在26 kDa處產生一明顯條帶，其大小與 Ecc 71菌株之HSL synthase相符。具HSL synthase基因之ECEXP-1選殖株與軟腐細菌Ecc及E. chrysanthemi (Ech)共同培養後，利用Thiobarbituric acid ( TBA )之方法進行果膠分解酵素活性之測定，結果顯示選殖株ECEXP-1可促進軟腐細菌Ecc與Ech之pectate lyase的產生，而僅對Ecc之polygalacturonase產生具促進效果；於crystal violet pectate ( CVP )培養基上亦顯示ECEXP-1可誘導Ecc及Ech在低濃度下產生大量果膠分解酵素而使CVP培養基提早凹陷。本研究利用農桿菌轉殖法( Agrobacterium-mediated transformation )將選殖之HSL synthase基因轉殖至台菸五號菸草，共計得到11個HSL synthase基因轉殖菸草品系。以cauliflower mosaic virus ( CaMV)之 35S promoter 及HSL synthase基因所設計之專一性引子對35S-PF2 / Hsl I-PR 應用PCR測試菸草轉殖品系，結果均可增幅出一大小約770 bp之DNA片段，顯示已成功將HSL synthase基因轉殖至菸草中；以HSL synthase基因轉殖菸草之RNA為模板進行反轉錄聚合酶鏈鎖反應 (reverse transcript polymerase chain reaction , RT-PCR )，均可增幅出一大小為470 bp之cDNA片段，顯示HSL synthase基因於菸草內可轉錄為messanger RNA；以離葉接種方式 ( detached leaf assay)將軟腐細菌接種於轉殖菸草品系，所有HSL synthase轉殖基因菸草品系對Ecc感受性皆較野生型菸草或pBI121載體轉殖菸草為低；而僅3個轉殖菸草品系對Ech之感受性與野生型菸草或pBI121載體轉殖菸草為低。

Quorum sensing is a system which bacteria monitor their own population densities and regulate genes expression through sensing the levels of signal molecules called autoinducers. Quorum sensing bacteria produce and release chemical signal molecules that increase in concentration as a function of cell density. The most common autoinducers in gram negative bacteria is acylated homoserine lactone (HSL) synthesized by HSL synthase gene. In this study, we cloned and characterized HSL synthase gene from Erwinia carotovora subsp. carotovora ( Ecc ) ZL1 strain and the HSL synthase gene transgenic tobacco were generated. The susceptiblity of the HSL synthase gene transgenic tobacco tissue to soft rot erwinia was examined. Primer pair expI-F / expI-R was designed according to the sequence of expI gene (the HSL synthase gene of Ecc SCC3193) to amplify the HSL synthase gene of Ecc strains from colored calla lily. A 1.2 kb DNA fragment was amplified from each of 20 Ecc strains by polymerase chain reaction (PCR). The 1.2 kb DNA fragment amplified from Ecc ZL1 was further cloned and sequenced. It showed 92% identities with hsl I gene of Ecc 71. HSL synthase gene from Ecc ZL1 was constructed into pT7-7 expression vector and transformed into Escherichia coli BL21 DE3. The total protein profiles of E. coli BLEXP-1 carrying the HSL synthase gene from Ecc ZL1 were assayed by Sodium dodecyl sulfate polyacrylamide gel electrophoroesis (SDS-PAGE). It showed that a 26 kDa protein band was observed. In addition, E. coli ECEXP-1 could accelerate the pit formation by Ecc ZL1 and Ech CAS11 on crystal violet polypectate plate (CVP). Coincubation of high densities of E. coli ECEXP-1 carrying HSL synthase gene from Ecc ZL1 with low densities ( 106 CFU/ml) culture Ecc ZL1 could enhance the production of pectate lyase and polygalacturonase, whereas coincubation of E. coli ECEXP-1 with Ech CAS11 only enhance the production of pectate lyase but not polygalacturonase. A total of 11 lines of HSL synthase gene transgenic tobacco were generated by Agrobacterium-mediated transformation technique. Expression of HSL synthase gene in each of transgenic tobacco lines was confirmed by reverse transcript polymerase chain reaction (RT-PCR) with primer pair 35S-PF2 / hsl I-PR. The susceptibility of transgenic tobacco leaf tissues to soft rot Erwinia was assayed. The results revealed that leaf tissues of all the transgenic tobacco lines were less susceptiblie to Ecc ZL1 and ZL9 than that of wild type or vector transgenic tobacco leaf tissues. Whereas only three tobacco lines were less susceptible to Ech CAS7 and CAS11 than that of wild type or vector transgenic tobacco.