秋茄硫氧还蛋白调控活性氧平衡增强烟草耐盐机制研究  

Overexpression of KcTrxf in tobacco enhances salt tolerance through the regulation of ROS homeostasis under NaCl stress

作　　者：荆晓姝 孙苑玲 向敏[2] 钱泽勇 郎涛[1] 赵瑞[1] 沈昕[1] 陈少良[1] 

机构地区：[1]北京林业大学生物科学与技术学院 [2]海南师范大学生命科学学院

出　　处：《北京林业大学学报》2015年第6期17-26,共10页Journal of Beijing Forestry University

基　　金：国家自然科学基金项目(31270654;31160150);教育部科学技术研究(科学技术类)项目(113013A);人事部留学人员科技活动项目(2012001);高等学校学科创新引智计划项目(111 Project;B13007);教育部创新团队发展计划项目(IRT13047)

摘　　要：硫氧还蛋白(Trxs)能调控细胞的氧化还原状态,在木本植物中Trxs与耐盐性的关系尚未研究。本文克隆了非泌盐红树秋茄的硫氧还蛋白基因KcTrxf,并研究KcTrxf在植物耐盐性中的作用。qRT-PCR结果显示,秋茄在盐胁迫下KcTrxf表达量上调,并且叶片中的非蛋白巯基(NPTs)的含量上升。KcTrxf基因的开放阅读框(ORF)长585bp,编码194个氨基酸,是一类定位于叶绿体中的f类硫氧还蛋白。将重组的35S:KcTrxf表达载体转入模式植物烟草中进行耐盐性分析,结果表明,KcTrxf提高了烟草的耐盐性。NaCl处理下,野生型烟草叶片中膜质氧化,并且积累大量活性氧,使叶绿素含量以及叶绿素a/b比值明显下降。转基因烟草一方面通过提高过氧化氢酶(CAT)以及抗坏血酸过氧化物酶(APX)的活性来清除H2O2,另一方面通过调节抗坏血酸-谷胱甘肽循环中(AsA-GSH cycle)的关键酶单脱氧抗坏血酸还原酶(MDAR)以及谷胱甘肽还原酶(GR)的活性来增加还原型谷胱甘肽水平,同时,还增加了叶片中非蛋白巯基的含量,进而清除活性氧,减少盐害引起氧化胁迫。因此,盐胁迫下转基因烟草中的叶绿素含量以及叶绿素a/b维持较高水平,从而维持较高的光合速率和生长状态。Thioredoxins （Trxs）play a crucial role in mediating the redox state in living cells; however, little is known how Trxs mediate salt tolerance in woody species. The aim of this study was to clone putative Trxf from non-secretor mangrove Kandelia candel and analyze its role in NaCl tolerance. Salt treatment enhanced NPTs（non-protein thiols）contents and increased transcription of Trxf in K. candel. The KcTrxf gene contained an open reading frame of 585 bp encoding a putative thioredoxin protein with 194 amino acids. Sequence and localization analyses revealed that KcTrxf is a typical thioredoxin in the chloroplast.To clarify the role of KcTrxf in salinity tolerance, it was transferred to tobacco, a model experimentalsystem for functional genetics. KcTrxf-transgenic lines were more salt tolerant than wild-type （WT） tobacco. In the WT tobacco, salinity（100 mmol/L NaCl）resulted in a marked reduction of chlorophyll content, and chlorophyll a/b ratio. The salt damage in WT resulted presumably from lipid peroxidation and ROS accumulation. However, the salt treatment caused less damage in KcTrxf-transgenic plants because of the up-regulated activity of catalase and ascorbate peroxidase. Moreover, KcTrxf-transgenic plants were able to increase the activities of MDAR （monodehydroascorbate reductase） and GR（glutathione reductase） in chloroplast AsA-GSH cycle, leading to an increase in the ratio of reduced glutathione（GSH）to oxidized glutathion（GSSG）and NPTs in the leaves. Therefore, KcTrxf-transgenic plants could better scavenge the salt-elicited reactive oxygen species（ROS）in leaf cells, compared to WT plants. In this way, reduction of chlorophyll content, and chlorophyll a/b ratio of KcTrxf-transgenic plants was consequently less restricted. As a result, KcTrxf-transgenic plants maintain photosynthesis and growth under salinity in the longer term.

关 键 词：红树 秋茄 硫氧还蛋白 过氧化氢酶 抗坏血酸过氧化物酶 AsA.GSH循环 NaCl胁迫 

分 类 号：S718.43[农业科学—林学] Q943.2[生物学—植物学]