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標題: 以蛋白質交互作用分析阿拉伯芥之Rab8A蛋白質及其參與之農桿菌感染過程
A protein interaction study of the Arabidopsis Rab8A protein and its involvement of Agrobacterium infection process
作者: Yin-Tzu Liu
關鍵字: 農桿菌;Rab8A蛋白質;Agrobacterium;Rab8A
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The Rab (Ras-like proteins in brain) protein, one of the small GTPase families, is common in eukaryotes and is primarily responsible for the endomembrane trafficking in plants. The Rab proteins include RabA-H subfamilies. The AtRabE (Rab8) subfamily is mainly involved in the post-Golgi transport to the plasma membrane in plant cells. Based on the previous studies, the AtRab8A, AtRab8B and AtRab8D' interacted with the Agrobacterium tumefaciens VirB2 (virulence) protein and Arabidopsis proteins AtRTNLB1, AtRTNLB2 and AtRTNLB4 in yeast two-hybrid assays. The RTNLB (reticulon-like B class) protein is located in the endoplasmic reticulum, and mediate tubular structure formations in plant cells. In this study, the glutathione S-transferase (GST) pull-down assays were used to detect interactions between AtRab and AtRTNLB proteins in vitro. The GST pull-down assay results showed that AtRab8A and AtRab8D' proteins interacted not only with A. tumefaciens virulence proteins VirB2, but also interacted with Arabidopsis proteins AtRTNLB1-4 and AtRTNLB8. In order to determine interaction strengths between AtRab8A, AtRab8B, AtRab8D' and AtRTNLB1-4, AtRTNLB8 proteins, the β-galactosidase enzyme activity assay in the yeast two-hybrid system were utilized. The yeast two-hybrid assay results showed that β-galactosidase enzyme activities were highest with VirB2 and AtRTNLB4. Among the tested yeast two-hybrid interactions between AtRab8A-8E and five AtRTNLB, AtRTNLB1-4, AtRTNLB8 proteins, the AtRTNLB4 with either the AtRab8A, AtRab8B, or AtRab8D' showed highest the β-galactosidase enzyme activity. Among the tested yeast two-hybrid interactions between different AtRTNLB proteins, the AtRTNLB2 with either the AtRTNLB2, AtRTNLB3, or AtRTNLB4 showed the highest β-galactosidase enzyme activity. To understand how the AtRab8A protein involved in A. tumefaciens infection process, the over-expression of AtRab8A or T7-tagged-AtRab8A transgenic plants were obtained and characterized. The transient transformation efficiencies and tumorigenesis efficiencies were higher in the AtRab8A over-expression transgenic plants than in the wild-type (Wassilewskija), suggesting that the AtRab8A protein may participate the A. tumefaciens infection process. Based on the results shown in this study and in previous studies, the AtRab8A protein may interact with VirB2, AtRTNLB1-4, and AtRTNLB8 proteins, and therefore participate A. tumefaciens infection process in plants.

Rab (Ras-like proteins in brain)為small GTPase的家族成員之一,普遍存在於真核生物中,在植物的Rab蛋白質主要負責細胞內的內膜運輸, 且可以再細分成A-H次家族,其中AtRabE (Rab8)次家族在植物細胞中主要可參與高基氏體到細胞膜之間物質的運送。且利用酵母菌雙雜合系統已發現AtRab8A、AtRab8B和AtRab8D'可與農桿菌致病蛋白質VirB2及植物蛋白質AtRTNLB1、AtRTNLB2和AtRTNLB4結合。而RTNLB為植物細胞內的reticulon-like 蛋白質,位於內質網上且幫助內質網管狀膜構造的形成。因此,本研究再進一步利用榖胱甘?硫轉移?蛋白質沉澱實驗(glutathione S-transferase [GST] pull-down assay)的方式檢測,在in vitro環境下AtRab和AtRTNLB蛋白質之間的交互作用,結果得知AtRab8A和AtRab8D'可與農桿菌中VirB2致病蛋白質結合,也可與植物蛋白質AtRTNLB1至4和AtRTNLB8結合。為了進一步瞭解AtRab8A、AtRab8B、AtRab8D'和VirB2及AtRTNLB1至4、AtRTNLB8蛋白質之間的結合力的差異,將上述有相互結合的蛋白質,利用酵母菌雙雜合系統並以β半乳醣甘?(β-galactosidase)酵素活性分析二測試蛋白質之間的結合能力。AtRab8A、AtRab8B、AtRab8D'蛋白質與VirB2的結合能力無顯著差異;而AtRTNLB蛋白質中,則以AtRTNLB4蛋白質與VirB2的結合能力最強。若檢測AtRab8和5種AtRTNLB之間的結合能力,可發現AtRab8A、AtRab8B、AtRab8D'與AtRTNLB4蛋白質的結合能力最強。而觀察五種AtRTNLB蛋白質兩兩之間的結合能力,可發現到AtRTNLB2、AtRTNLB3、AtRTNLB4與AtRTNLB2蛋白質的結合能力最強。此外,為了進一步瞭解AtRab8A蛋白質與VirB2和AtRTNLB蛋白質結合後,在植物中所扮演之功能。本研究建構了大量表現AtRab8A蛋白質的轉殖株和大量表現T7-tagged-AtRab8A重組蛋白質的轉殖株,來瞭解當轉殖株中大量表現AtRab8A蛋白質時,是否會影響轉殖株被農桿菌感染的效率。實驗結果顯示當大量表現AtRab8A蛋白質或T7-tagged-AtRab8A重組蛋白質時,其轉殖株被農桿菌感染後,短暫表現T-DNA的能力及腫瘤產生的效率都比野生株(Wassilewskija,Ws)高,可推測AtRab8A蛋白質可能參與農桿菌感染植物的過程。綜合本研究結果及前人研究,推測AtRab8A蛋白質可藉由與農桿菌VirB2致病蛋白質和植物蛋白質AtRTNLB1至4和AtRTNLB8結合後,進而參與農桿菌感染植物的過程。
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