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標題: 阿拉伯芥AtRab8蛋白質家族於土壤農桿菌感染植物過程之功能分析
Functional studies of the AtRab8 family in Agrobacterium-mediated plant transformation process
作者: 毓, 盧
Lu, Yu
關鍵字: Agrobacterium;阿拉伯芥;Arabidopsis;Rab;農桿菌;Rab
出版社: 生命科學系所
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農桿菌(Agrobacterium tumefaciens)是一種存在於土壤中的伺機性植物病原菌。其菌體內含有一特殊質體tumor-inducing plasmid (Ti plasmid),使農桿菌具備跨越“界”(kingdom)而能轉移遺傳物質的能力,故成為目前最廣泛應用於植物基因轉殖技術的工具之一。Ti質體內具有一段特殊的transfer DNA (T-DNA),及一系列與感染過程相關之重要致病基因(virulence gene,vir)。Vir蛋白質可參與T-DNA的產生、轉移、進入及鑲嵌進入植物細胞染色體之過程。且其中的virB操縱子(virB operon)所編碼產生的11種VirB蛋白質及VirD4蛋白質,共同組成一穿膜的第四型分泌系統(type IV secretion system,T4SS )。此系統負責將T-DNA及Vir蛋白質經由T4SS的特殊構造T-線毛(T-pilus)傳送至宿主細胞。T-線毛主要由VirB2及兩種次要蛋白質VirB5和VirB7所組成。已知在酵母菌雙雜合系統中,VirB2蛋白質可與AtRab8B結合,為了進一步了解AtRab8B及AtRab8家族中其他成員是否也有參與農桿菌感染植物之過程。本研究利用大量表現AtRab8B及在AtRab8B之N端分別加上T7-tag或His-tag的阿拉伯芥轉殖株,及AtRab8A、AtRab8C、AtRab8D、AtRab8E四種基因的突變株,檢測其受農桿菌感染的效率,以了解AtRab8各蛋白質在農桿菌感染過程中之角色。結果顯示大量表現AtRab8B轉殖株能提高被農桿菌轉殖的效率,而rab8A及rab8C突變株被農桿菌感染的效率下降,rab8D突變株則提高了產生腫瘤之效率,rab8E突變株則與野生株無明顯的差異。由此推測AtRab8A、AtRab8B、AtRab8C、AtRab8D皆極有可能能參與農桿菌轉殖植物的過程。為了瞭解AtRab8家族各成員,在阿拉伯芥的生長發育過程可能扮演的角色。故本研究使用逆轉錄聚合酶連鎖反應,及阿拉伯芥生物晶片之資料庫內所整合的實驗結果,得知AtRab8各成員在阿拉伯芥不同生長階段、組織部位及受到各種逆境時的表現量各有不同。顯示各個AtRab8可能在阿拉伯芥生長發育過程扮演重要角色,且在不同組織部位中具有不同生理功能。

Agrobacterium tumefaciens exists in soil and is a plant pathogen. It has a unique plasmid, called tumor-inducing plasmid (Ti plasmid), which contains a transfer DNA (T-DNA) region and several virulence genes. During Agrobacterium infections, the T-DNA is transferred from the bacterium into plant cells where it integrates into plant genome. The expressions of several virulence genes in bacterium will help the T-DNA production, transfer and integration. A. tumefaciens has become a widely useful tool to generate genetic modified crops because of its ability to genetically transform a wide variety of plants. A. tumefaciens utilizes a Type IV secretion system (T4SS) to transfer T-DNA and virulence proteins to plants. The T4SS is composed of two major structural components: the T-pilus and a membrane associated complex that is responsible for translocating substrates across both bacterial membranes. The VirB2 protein is the major component of the T-pilus. A Previous study showed that VirB2 interacts with AtRab8B in the yeast two-hybrid system. In order to understand if the AtRab8B and other members of the AtRab8 gene family participate in the A. tumefaciens infection process, we examined the transformation efficiencies of the over-expression AtRab8B transgenic plants and rab8A, rab8C, rab8D, and rab8E T-DNA insertion mutants. Over-expressions of AtRAB8B proteins in transgenic plants results in plants that are hyper-susceptible to Agrobacterium-mediated transformation. The rab8A and rab8C mutants show reduced levels of Agrobacterium-mediated root transformation, whereas the rab8D mutant shows higher transformation efficiency. These results suggest that AtRab8A, B, C, and D are possibly involved in A. tumefaciens infection process. Furthermore, results from RT-PCR (reverse transcription polymerase chain reaction) and microarray analyses demonstrated that each member of the AtRab8 gene family ubiquitously express in various plant tissues and is up or down-regulated under different stresses, suggesting each AtRab8 may play important roles in plants.
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