白藜受菜豆細菌性斑點病菌誘導過敏性反應過程中Rubisco活化酵素基因之表現

Expression of rca Gene in Chenopodium quinoa During the Hypersensitive Response Induced by Pseudomonas syringae pv. Syringae

蔣本慈( B. T Chiang) ； 黃秀珍( H. C Huang)

《植物病理學會刊》 12卷2期 (2003/06) Pp. 93-102

https://doi.org/10.6649/PPB.200306_12(2).0004

摘要

白藜(Chenopodium quinoa)受到竹嵌紋病毒(Bamboo mosaic virus ；BaMV-S)或菜豆細菌性斑點病菌(Pseudomonas syringae pv. Syringar strain 61.Pss61)感灣時，會被誘導產生過敏性反應。為探討竹嵌紋病毒所誘導的白藜過敏性反應之機制，曾使用cDNA subtraction方式選殖經病毒誘導過敏反應後第三天表現的基因，選殖到pTA2-9之選殖株，其所含的插入片段序列乞含有部分的ribulose-1,5-bisphosphate carboxylase/oxygenase活化酵素，(Rubisco activase, rca)基因序列。進一步以5'及3'RACE選殖得到1809bp長片段，包括5端121bp的非轉譯序列、1314bp轉譯區及3端374bp之非轉譯序列。而轉譯區可轉譯出含438個氨基酸的蛋白質、分子量為47.8kDa、等電點(pI)為6.875，此胺基酸序列與同為藜科的菠菜RCA蛋白最為相似。RCA蛋白為光合作用暗反應中，固定二氧化碳的酵素Rubisco的活化子，經南方雜合術分析確定rca以單套基因形式存在於白藜基因體內。以水稻的RCA蛋白之血清進行西方墨點術分析及利用reverse transcription-polymerase chain reaction(RT-PCR)方式分析，結果顯示白藜葉內含有L及S型的RCA蛋白，但以S型含量較高。依據rcu的cDNA衍生氨基酸序列推論，本研究所選殖到的rca之cDNA蛋白所轉譯的蛋白質屬ｓ型，又將此基因在大陽桿菌中以T7-RNA ploymerase depemdent系統表現，並以西方墨點術確認此蛋白質為S型。在Pss61所誘發的過敏性反應過程中，其mRNA的表現量於第6-9小時後開始有顯著下降的趉勢，約為對照組rca mRNA的表現量的0.98至0.55倍。而以西方墨點術分析顯示白藜在經Pss61所誘導產生之過敏性反應中，RCA蛋白質的累積量與對照組相比較並無顯著差異，顯示此蛋白質可能很穩定，而rca mRNA累積量有下降的趨勢，可能是因過敏反應引發植物細胞生理代謝改變後之基因調控結果。

關鍵字

白蔾 ； 過敏性反應 ； Rubisco活化酵素 ； 菜豆細菌性斑點病菌

並列摘要

A full-length rca cDNA encoding Rubisco activase was isolated from leaf tissues of Chenopodium quinoa using subtractive hybridization and following 5' and 3' RACE-PCR cloning. This clone comprises 1809 bp, consisting of a 121-bp 5'unstralated region, a 1314 bp coding sequence of rca cDNA, and 374-bp 3'unstralated region. The deduced sequence of RCA includes 438 amino acid residues of Mr 47.8 kDa and pI 6.875. A single copy of rca gene is detected in the genome of C. quinoa based on Southern blot assay. According to western blot analysis with a rice RCA serum and RT-PCR experiment, the results indicated that there are two isoforms of RCA protein present in C. quinoa and the S-form is abundant. The clone isolated here encodes S-form RCA. Furthermore, the northern blot assay showed that the rca mRNA expression of C. quinoa declined 6-9 hr after the hypersensitive response (HR) induced by Pseudomonas syringae pv. syringae strain Pss61. However, the accumulation of RCA protein during the HR was the same based on the western blot analysis.

並列關鍵字

Chenopodium quinoa ； hypersensitive response ； Rubisco activase ； Pseudomonas syringae pv. syringae strain Pss61