在阿拉伯芥大量提升耐熱古生菌Sulfolobus solfataricus 硫氧化還原系統對非生物性逆境抗性之研究

Abstract

硫氧化還原蛋白（Thioredoxin, 簡稱Trx）是一類利用硫基（Thiol group）氧化還原反應（2SH←→S-S）調節細胞氧化還原狀態的蛋白，與其專一性的Trx還原酶（The NADPH-dependent thioredoxin reductase, 簡稱NTR）構成NTR/Trx系統，在植物氧化逆境抗性反應中扮演重要的角色。Sulfolobus solfataricus是一種生長在極端環境的古生菌，為了解此系統對植物逆境抗性之影響，本研究將S. solfataricus 兩個Trx基因SsTrxA1、SsTrxA2，以及一個NTR基因SsTrxB3，分別利用35S啟動子驅動構築成可在植物大量表現的質粒，包括OE-SsTrxA1、OE-SsTrxA2、OE-SsTrxB3 、OE- SsTrxA1+ SsTrxB3、OE- SsTrxA2+SsTrxB3等5個質粒，並利用農桿菌轉殖大量表現在阿拉伯芥，以分析轉殖植物對逆境之抗性。RT-PCR分析結果顯示，五種質粒均可成功的大量表現在阿拉伯芥植株中，各基因構築均可獲得6個以上轉殖系。轉殖株經過鎘、巴拉刈處理後，顯示SsTrxB3 與 SsTrxA2+SsTrxB3轉殖株有較佳的逆境抗性，在逆境下與未轉殖植株比較，有較長的根長與較大的鮮重。此外，熱逆境分析結果顯示OE- SsTrxA2B3具有較佳的先天耐熱性。這些結果顯示來自S. solfataricus的NTR/Trx系統能提高植物的逆境抗性，未來應具有潛力應用於增加作物耐多重逆境抗性。

NADPH-dependent thioredoxin reductase/Thioredoxin (NTR/Trx) system catalyzes disulfide-bond reduction to regulate the redox status of cells, ubiquitously exists in all organisms and involves in oxidative stress response. The NTR/Trx system of a hyperthermophilic archaeon, Sulfolobus solfataricus, has been found to play a major role in its extremely thermal tolerance. To study the impact of S. solfataricus NTR/Trx system on plant abiotic stress tolerance, S. solfataricus NTR/Trx system genes (two Trx genes：SsTrxA1, SsTrxA2 and one NTR gene ：SsTrxB3) were solely or simultaneously overexpressed in Arabidopsis thaliana to evaluate their roles in abiotic stress tolerance. Five plasmids including SsTrxA1, SsTrxA2, SsTrxB3, SsTrxA1+ SsTrxB3 and SsTrxA2+SsTrxB3 under the control of a CaMV 35S promoter were constructed and transformed to Arabidopsis by Agrobacterium tumefaciens. At least 6 transgenic lines were obtained for each construct and most of them showed highly accumulation of NTR or Trx mRNA. Functional analysis indicated that OE-SsTrxB3 and OE-SsTrxA2B3 plants exhibited enhanced tolerance of transgenic plants to Cd and paraquat.Moreover, SsTrxA2+SsTrxB3 plants also showed increased thermal tolerance. Taken together, the S. solfataricus NTR/Trx system is able to enhance stress tolerance in Arabidopsis. This system may be applied for the development of new strategies to enhance crop tolerance to multiple stresses.