持續大量表現AtMsrB7於阿拉伯芥可增進植物對細菌性病原菌與氧化逆境之耐受度

Overexpression of methionine sulfoxide reductase B7 (AtMsrB7) in Arabidopsis enhances tolerance to pathogen infection and oxidative stress

李書宏(Shu-Hong Lee)

指導教授 ： 王玉麒

國立臺灣師範大學/理學院/生命科學研究所/碩士(2009年)

摘要

前人研究顯示轉殖甜椒的PFLP (plant ferredoxin-like protein) 基因，可以提昇轉殖植物對於軟腐病的抗性 (You et al., 2003)。本實驗室尚未發表的阿拉伯芥Affymatrix microarray資料則顯示，感染軟腐病菌 (Erwinia carotovora ssp. carotovora, Ecc) 之阿拉伯芥PFLP轉殖株內，MsrB7 (methionine sulfoxide reductase B7) 基因的表現量增加約10倍。本研究進一步對感染Ecc的野生型阿拉伯芥植株 (WT) 進行定量RT-PCR (Quantitative reverse transcription polymerase chain reaction, q-RT-PCR) 分析，結果也發現MsrB7基因在WT中也因Ecc感染而增加表現，本研究於是推測MsrB7可能參與植物的抗病反應機制。為驗証前述的推測，本研究選殖阿拉伯芥的MsrB7 (AtMsrB7) 基因，並藉CaMV35S啟動子之驅動，使原本主要表現於根部的MsrB7，持續大量表現於阿拉伯芥全株中，並對轉殖植株進行抗病能力的檢測。基因轉殖實驗結果顯示MsrB7轉殖株 (MsrB7OX) 對於Ecc的感染與殺草劑巴拉刈 (Methyl viologen, MV) 的處理都明顯增加耐受度，經由DAB (3,3-diamino-benzidine) 的染色結果可知，MsrB7OX轉植株內的H2O2累積量較WT對照組為低，顯示轉植株內移除ROS (reactive oxygen species) 的活性增高。比較三種ROS移除相關酵素活性的實驗結果顯示，MsrB7OX轉植株內peroxidase和thioredoxin reductase的活性明顯提升，而catalase的活性則無顯著變化。本研究也利用q-RT-PCR檢測轉殖株內各種已知與抗病相關基因的表現情形，結果發現MsrB7轉植株中，SA-dependent pathway上的基因包括NPR1、WRKY70與PR1等的表現量均有顯著增加。綜合上述實驗結果推測，持續表現MsrB7可增加植株對Ecc耐性之原因可能是藉由移除過多ROS與活化SA-dependent pathway抗病機制來達成。另外，本研究也發現轉殖株除了能耐受Ecc的處理之外，亦能耐受青枯病菌 (Ralstonia solanacearum) 的感染。因此持續表現MsrB7基因，可提高植物對氧化逆境及至少兩種細菌病害的多重耐性。

關鍵字

阿拉伯芥 ； 軟腐病 ； 青枯病 ； 氧化逆境 ； 活性氧分子

並列摘要

Previous studies showed that Arabidopsis transformed with sweet pepper ferredoxin-like protein (PFLP) gene exhibited a resistance to Ecc (Erwinia carotovora ssp. carotovora) infection (You et al., 2003). Subsequent unpublished microarray analyses of theses transgenic plants found the transcripts of methionine sulfoxide reductase B7 (AtMsrB7) increased over ten folds upon Ecc infection. Our results of quantitative reverse transcription-polymerase chain reaction (q-RT-PCR) analysis to wild-type Arabidopsis had showed that MsrB7 transcript was primarily present in the roots and its expression would significantly increase after bacterial challenge. To investigate the function of MsrB7, transgenic Arabidopsis plants overexpressing MsrB7 (MsrB7OX) have been generated, and these transformants did exhibit substantial tolerance to Ecc infection. Moroever, DAB staining revealed that after pathogen and methyl violgen (MV) treatment, MsrB7OX plants accumulated less hydrogen peroxide than the wild type. Two ROS-removal enzymes, including peroxidase and thioredoxin reductase, displayed higher activities in MsrB7OX than that in the wild-type plants. Furthermore, several genes involved in the SA-dependent pathway, such as NPR1, WRKY70, and PR1 were up-regulated in the MsrB7OX plants from q-RT-PCR analysis . According to these results, we suggest that overexpression of MsrB7 in Arabidopsis might enhance the tolerance to Ecc infection via elimination of the hyperproduction of ROS and activation of the SA-dependent pathway in plant defense mechanism. We also observed that MsrB7OX could resist Ralstonia solanacearum infection. Thus, our data suggest that overexpression of MsrB7 in Arabidopsis could enhance transfenic plants tolerance to oxidative stress, and lead to the tolerance to the infection of at least two bacterial pathogens.

並列關鍵字

Arabidopsis ； Methionine sulfoxide reductase B (MsrB) ； Methyl viologen ； Erwinia carotovora ssp. carotovora (Ecc) ； reactive oxygen species (ROS) ； Ralstonia solanacearum

參考文獻

Abramovitch, R.B., Anderson, J.C., and Martin, G.B. (2006). Bacterial elicitation and evasion of plant innate immunity. Nature Reviews 7, 601-611.

Bostock, R.M. (2005). Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annual Review of Phytopathology 43, 545-580.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248-254.

Broekaert, W.F., Delaure, S.L., De Bolle, M.F., and Cammue, B.P. (2006). The role of ethylene in host-pathogen interactions. Annual Review of Phytopathology 44, 393-416.

Cabreiro, F., Picot, C.R., Friguet, B., and Petropoulos, I. (2006). Methionine sulfoxide reductases: relevance to aging and protection against oxidative stress. Annals of the New York Academy of Sciences 1067, 37-44.