受高溫影響之文心蘭葉綠體型銅鋅超氧歧化酶基因表現之分析

Functional Analysis of a Chloroplast Copper/Zinc Superoxide Dismutase of Oncidium Gower Ramsey under Heat Stress

吳怡珊(Yi-Shan Wu)

指導教授 ： 王淑美

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.10261

摘要

文心蘭在夏季高溫的環境下，可縮短花芽發育期，但花序之品質卻明顯降低，花軸分叉數和總花朵數都顯著減少，部分花朵亦有畸形的現象，且花軸長度也明顯短於在常溫下生長的花序。迄今我們對於影響文心蘭花序品質的機制毫無所知。為期改善夏季高溫影響花序發育的情形，本論文選殖在高溫下影響文心蘭花軸品質之相關基因，做為日後研究之基礎。實驗採用文心蘭（Oncidium）切花品種南西（Gower Ramsey）三年株齡之植物個體為材，分別培養在適溫（28/23℃）及高溫（35/30℃）之下，然後取其花軸尖端部位，利用DDRT-PCR（differential display reverse transcription PCR）方法，選殖獲得298個基因建構體，經定序比對，再從中挑選與細胞氧化逆境相關之基因具有高度相似性者，期望以轉殖株測試轉殖之基因在高溫下的生理功能。針對數個與氧化逆境相關的基因（BEAM09、BFAM09、BHGM05、BHGM22、BIAM14）經由5’RACE (rapid amplification of cDNA ends)的技術得到文心蘭3個基因lipase (class3) (Olipase)、cysteine synthase (OOASTL)及chloroplast Cu-Zn superoxide dismutase(OSOD)之cDNA序列，進一步將HSP101:: OSOD及HSP101:: antiOSOD構築於pBI101中轉殖至阿拉伯芥，連同阿拉伯芥葉綠體型CuZnSOD(csd2)突變株一同觀察高溫逆境下的基因表現和外表型態。另外，依不同型態的SOD對於抑制劑的敏感性和電泳中移動速率之快慢來鑑定SOD同功酶，可知文心蘭至少有四個CuZnSOD、一個FeSOD和一個MnSOD，這六個SOD同功酶在文心蘭不同組織部位的表現強弱也不盡相同。即使文心蘭和阿拉伯芥的葉綠體型CuZnSOD都會受到高溫逆境的誘導，但是高溫仍會對文心蘭和阿拉伯芥造成光氧化逆境傷害。CSD2突變株意外地有較野生型高的CSD2表現，然而文心蘭SOD或CSD2在高溫環境下阿拉伯芥中的表現強弱雖然對於植株外表型態沒有直接的影響，但是可有助於抵抗部分的光氧化逆境，並延緩高溫對阿拉伯芥所造成的傷害。對於阿拉伯芥或文心蘭在高溫所表現的花序差異目前沒有詳細的研究報導，本文初步探討受高溫影響之文心蘭花序品質的相關基因及文心蘭SOD在高溫逆境可能參與的角色，影響文心蘭花序品質的詳細機制尚待進一步的研究。

關鍵字

氧化逆境 ； 活性氧 ； 超氧歧化酶

並列摘要

Oncidium Gower Ramsey is one of major cut flower species in Taiwan, and the flower production is all over the year. However, high temperature of summer and autumn results in the poor quality of cut flowers, by that not only the branching and floral number of inflorescence drop severely, but the length of inflorescence is shorter than that developed in cool seasons. In order to understand high temperature-related genes in inflorescence of Oncidium, DDRT-PCR technique was used. 298 gene fragments shown differential expression were cloned and sequenced. After Blast alignment, six clones possessing high homology with known genes related to oxidative stress were selected. Among several oxidative stress related genes, three complete cDNA sequences coding for Oncidium lipase(Olipase)、cysteine synthase(OOASTL) and chloroplast CuZnSOD(OSOD) were cloned by 5’RACE. For high temperature stress, AtHSP101 promoter were used to drive sense and antisense OSOD in Arabidopsis thaliana. Even though OSOD and CSD2 (chloroplast CuZnSOD of Arabidopsis) were induced by high temperature, photooxidative stress still occurred in Oncidium and Arabidopsis. csd2 mutant unexpectedly had higher expression of CSD2 than wild type, and had more branch number in both moderate and high growth temperature(35℃). The phenotype of Arabidopsis is not directly affected by OSOD or CSD2, but the enhancement of the active oxygen-scavenging system that leads to increased oxidative stress protection and delayed the harm caused by high temperature in transgenic plants.

並列關鍵字

ROS ； SOD ； heat stress

參考文獻

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