马尾松谷胱甘肽过氧化物酶PmGPX6基因cDNA克隆及转化拟南芥耐旱性初步研究  

Cloning of Glutathione Peroxidase Pm GPX6 Gene from Pinus massoniana and the Study on Drought Tolerance of Transgenic Arabidopsis thaliana

作　　者：蔡琼[1,2] 丁贵杰[1,2] 文晓鹏[3] CAI Qiong DING Gui-jie WEN Xiao-peng(Guizhou Institute for Forest Resources ＆ Environment, Guizhou University, Guiyang 550025, Guizhou, Chin College of Forestry, Guizhou University, Guiyang 550025, Guizhou, China Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou University, Guiyang $50025, Guizhou, China)

机构地区：[1]贵州省森林资源与环境研究中心,贵州贵阳550025 [2]贵州大学林学院,贵州贵阳550025 [3]贵州省农业生物工程重点实验室,贵州贵阳550025

出　　处：《林业科学研究》2016年第6期839-846,共8页Forest Research

基　　金：国家自然基金(31260183);国家863课题(2011AA10020301);贵州省重大专项(黔科合重大专项字[2012]6001号);国家科技支撑课题(2015BAD09B0102)

摘　　要：[目的]克隆马尾松谷胱甘肽过氧化物酶基因,并对其进行功能研究。[方法]采用RACE技术克隆基因c DNA序列,实时荧光定量PCR检测基因在马尾松干旱胁迫下的表达模式,花序浸泡法转化拟南芥,并对转基因与野生型拟南芥的生长表型和根系生长进行分析。利用荧光显微镜技术对转基因拟南芥不定根进行GFP荧光检测。[结果]克隆到1个871 bp的GPX基因全长c DNA序列,命名为Pm GPX6。Pm GPX6包括513 bp的完整开放阅读框,编码170个氨基酸残基。Pm GPX6蛋白与油松Pt GPX蛋白同源性达95%。Pm GPX6在马尾松根中高表达,茎、叶中表达量低。在干旱胁迫下,Pm GPX6在根、茎、叶中的表达量均在第15天达到最大,随后出现下降趋势。过表达Pm GPX6与野生型拟南芥植株在正常水分条件下表型与根长差异不大,但在干旱胁迫下,转基因植株根系更长。转基因拟南芥根在蓝色光激发下能发出强烈的绿色荧光,表明Pm GPX6基因能高效表达。[结论]推测Pm GPX6可能参与马尾松干旱胁迫应答。[Objective]To clone the glutathione peroxidase gene from Pinus massoniana and evaluate its gene functions. [Methods]The gene was cloned using RACE methods. Quantitative real-time PCR was used to analyze the gene expression in P. massoniana under drought stress. Transgenic Arabidopsis thaliana lines were obtained by dipping flowering plants. The phenotypes and root developments of wild-type and transgenic A. thaliana were evaluated. The activity of green fluorescence protein on root in transgenic A. thaliana plants was studied using fluorescence microscopy. [Results]The designated Pm GPX6 was 871 bp in length with an open reading frame（ 513 bp）,capable of encoding a predicted protein of 170 amino acids. The sequencing analysis indicated that the deduced amino acids shared 95% of identity with Pt CBL from Pinus tabulaeformis. The expression levels of Pm GPX6 were higher in roots than in stems and leaves,and the Pm GPX6 expression increased in the initial 15 days,and then de-creased under drought stress. No differences in the phenotypes and root lengths were observed between the wild-type and transgenic A. thaliana under normal conditions containing 0% PEG 6000. Nevertheless,the root lengths of transgenic A. thaliana were longer than those of the wild-type lines under drought stress. Bright green fluorescence was observed in transgenic A. thaliana under fluorescence microscopy,indicating that Pm GPX6 is expressed efficiently. [Conclusion]These findings indicate that the Pm GPX6 gene may play an important role under drought stress.

关 键 词：马尾松 谷胱甘肽过氧化物酶 转基因拟南芥 抗旱性 

分 类 号：S791.248[农业科学—林木遗传育种]