《植物生理学报》 2015, 51(1): 127-135

小桐子非特异性脂质转移蛋白A的基因克隆及其表达和功能分析

王海波^1,2, 邹竹荣¹, 龚明^1,*

¹云南师范大学生命科学学院, 生物能源持续开发利用教育部工程研究中心, 云南省生物质能与环境生物技术重点实验室, 昆明650500; ²曲靖师范学院生物资源与环境科学学院, 云南曲靖655011

通信作者：龚明；E-mail: gongming63@163.com；Tel: 0871-65516069

摘　要：

基于我们最近获得的小桐子低温驯化转录组和数字基因表达谱数据, 本工作研究了低温驯化条件下差异表达变化较大的非特异性脂质转移蛋白A基因JcnsLTPA。克隆到该基因的cDNA序列全长833 bp, 开放阅读框长度513 bp, 编码170个氨基酸, 存在ATT_LTSS典型保守功能基序。其启动子区域中鉴定到了TATA框、CAAT框、CATA框、W框等顺式作用元件以及CRT/DRE低温响应元件。半定量RT-PCR分析表明, 该基因在茎、根、叶中都有表达, 以茎中表达量最高、且受低温诱导最显著。同时, 酵母表达JcnsLTPA也提高了重组酵母菌的抗低温能力。这些结果充分说明了小桐子JcnsLTPA是与抗冷性密切相关的基因, 可以用于小桐子的抗冷性遗传改良。

关键词：小桐子; 非特异性脂质转移蛋白A; 基因克隆; 酵母表达

收稿：2014-11-21   修定：2014-12-16

资助：国家自然科学基金(31260064、31460059和31460179)和云南省教育厅科研基金重大专项(ZD2010004)。

Molecular Cloning, Expression Analysis and Functional Characterization of the Gene Encoding Non-Specific Lipid Transfer Protein A from Jatropha curcas

WANG Hai-Bo^1,2, ZOU Zhu-Rong¹, GONG Ming^1,*

¹School of Life Sciences, Yunnan Normal University, Engineering Research Center of Sustainable Development and Utilization of Biomass Energy, Ministry of Education, Key Laboratory of Biomass Energy and Environmental Biotechnology of Yunnan Province, Kunming 650500, China; ²College of Biological Resource and Environmental Science, Qujing Normal University, Qujing, Yunnan 655011, China

Corresponding author: GONG Ming; E-mail: gongming63@163.com; Tel: 0871-65516069

Abstract:

From our previous data of the transcriptome and digital gene expression profiling of cold-acclimated Jatropha curcas, a gene encoding the non-specific lipid transfer protein A (JcnsLTPA), with highly differential expression under chilling, was selectively studied herein. The full length of our cloned JcnsLTPA cDNA is 833 bp, with the entire open reading frame (ORF) of 513 bp encoding a polypeptide of 170 amino acids (with typical ATT_LTSS motif). Its promoter region was characterized by prediction with TATA-box, CAAT-box, CATA-box, W-box, and the cold responsive elements of CRT/DRE. Semi quantitative RT-PCR analysis revealed that JcnsLTPA was expressed in stem, root, and leaves, with abundant and remarkably chilling-induced expression particularly in stem. Meanwhile, the yeast expression of JcnsLTPA endued the recombinant yeast strain with an enhanced resistance to low temperature. Conclusively, these results largely implicated the cold relevance of JcnsLTPA gene that should be applicable for the genetic improvement on the cold tolerance of J. curcas.

Key words: Jatropha curcas; non-specific lipid transfer protein A; gene cloning; yeast expression