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結球白菜之銅/鋅超氧化歧化酶(Cu/Zn superoxide dismutase, SOD)基因與過氧化氫酶(Catalase, CAT)基因的選殖與分析

By 尤進欽, 曾夢蛟, Jinn-Chin Yiu and Menq-Jiau Tseng

Abstract

Superoxide dismutase (SODs)are metalloenzymes that are ubiquitous among aerobic organisms and are very efficient at scavenging O2 -.Hydrogen peroxide\ud (H2 O2)is synthesized as a byproduct of photorespiration﹐B-oxidation of fatty also as a consequence of biotic and abiotic stresses. Catralase(CAT)is the primary H2 O2\ud Scavenger in peroxisomes of leaves.\ud A cDNA library was constructed using poly A RNA from 800 ppb sulfur-dioxide-fumigated Chinese cabbage leaves for 7 hours. The cDNA library was screened for the sod and cat genes by using a maize sod4 and catl as probe. The insert of Psod62(sod62) has 754 bp deduces an open reading frame of 152 amino acids. The longest and complete inserts was found in Pcat78 of 1694 bp containing an open reading frame of 492 amion acids.\ud The sod62 and cat78 protein and SOD and CAT specific activity could be detected using SDS-PAGE by introduction of sod62 and cat78 into E.coil expression vector.\ud High levels of sod62 and cat78 Mrna could be detected in Chinese cabbage after 1 hour of 200 μM of MV treatment﹐and remained prominent thereafter till 24 hours.超氧化歧化酶(superoxide dismutase,SOD)廣泛而普遍的存在所有好氧的生物體內,可有效率地去除氧自由基。許多的生物性及升生物性逆境、光呼吸、脂肪酸β-氧化作用之中間產物都會產生過氧化氫(H202)危害植物。過氧化氫酶(catalase,CAT)能催化H2O2轉變成H2O及O2,是葉片過氧化盤中去除過氧化氫的最主要酵素。 \ud 本試驗以經800ppb二氧化硫(SO2)處理7小時之結球白菜(濱綠)葉片為材料,萃取其polyA RNA,構築cDNA庫。以玉米sod4及cat1基因為探針,篩選結球白菜之Cu/Zn-sod及cat基因。篩選的Sod62之核酸序列為754bp,可轉譯成具有一個152個胺基酸的open reading frame(ORF)。而cat78之核酸序列為1694bp,可轉譯成具492個胺基酸的ORF。\ud 將sod62及cat78基因分別構築至表現載體pQE32及pQE3l,並加入IPTG誘導使其大量表現,可在SDS-PAGE上55kDa及57kDa處有一明顯條帶。在nativePAGE上,有多出一SOD及CAT活性染色條帶出現。證實篩選之sod及cat基因可輯譯出SOD及CM蛋白質活性。結球白菜生長4週的葉片噴灑2OO M巴拉刈(MV),進行北方墨點分析之結果顯示,處理結球白菜1小時後,即可偵測到sod62 RNA表現,迨24小時仍持續增加。而CAT的表現情形,未處理前即有表現,但於處理MV l小時後,CAT的表現量顯著增加,而後也保持很高的表現量,迨24小時後仍持續表現

Topics: Chinese cabbage, Cu/Zn superoxide dismutase gene, Catalase gene, Cloning, 銅/鋅超氧化歧化酶基因, 過氧化氫酶基因, 選殖, 結球白菜
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81807
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