生物技术通报 ›› 2017, Vol. 33 ›› Issue (11): 84-91.doi: 10.13560/j.cnki.biotech.bull.1985.2017-0416

• 研究报告 • 上一篇    下一篇

木薯MeTPS9基因克隆及表达特性分析

丁泽红, 付莉莉, 铁韦韦, 颜彦, 胡伟   

  1. 中国热带农业科学院热带生物技术研究所,海口 571101
  • 收稿日期:2017-05-22 出版日期:2017-11-26 发布日期:2017-11-22
  • 作者简介:丁泽红,男,副研究员,研究方向:植物分子生物学;E-mail:dingzehong@itbb.org.cn
  • 基金资助:
    国家自然科学基金项目(31600198),海南省自然科学基金项目(20163120)

Clone and Expression Characteristics of MeTPS9 Gene in Cassava

DING Ze-hong, FU Li-li, TIE Wei-wei, YAN Yan, HU Wei   

  1. Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Science,Haikou 571101
  • Received:2017-05-22 Published:2017-11-26 Online:2017-11-22

摘要: 旨在揭示木薯MeTPS9基因在干旱、低温、遮荫等非生物胁迫应答中的作用。用RT-PCR的方法从木薯叶片中克隆MeTPS9基因(GenBank登录号:MF276889),用MEGA软件构建系统进化树,用DnaSP软件分析基因结构变异,用PlantCARE分析启动子元件,用荧光定量PCR技术分析MeTPS9在不同组织中以及响应不同非生物胁迫的表达特性。结果显示,从木薯中克隆了一个TPS基因MeTPS9。该基因具有2 598 bp的开放阅读框,编码865个氨基酸,含有TPS家族保守结构域。系统进化树分析表明,MeTPS9与荠菜花和拟南芥中同源基因的亲缘关系较近,序列相似性分别为76.2%和75.6%。基因结构变异发现,木薯野生种和栽培种之间共有66个突变位点,其中11个为错义突变。启动子元件分析表明,MeTPS9含有干旱相关元件MBS、低温相关元件LTR、热胁迫相关元件HSE、以及ABA相关元件ABRE。实时荧光定量PCR分析表明,MeTPS9的表达量在须根中最高,在叶片中最低。而且,MeTPS9基因的表达能被干旱、低温、和遮荫处理显著诱导。MeTPS9在转录水平对木薯干旱、低温、和遮荫胁迫起调控作用,可作为候选基因进一步研究其在木薯抗逆中的作用。

关键词: 木薯, MeTPS9, 海藻糖合成酶, 结构变异, 表达分析, 非生物胁迫

Abstract: This work aims to reveal the function of MeTPS9 gene in abiotic stresses(e.g.,drought,cold,and shade)in cassava. RT-PCR method was applied to clone MeTPS9 gene(GenBank accession number:MF276889)from cassava leaves,MEGA software to construct its phylogenetic tree,DnaSP software to analysze its structural variation,PlantCARE software to analyze its promoter cis-element,and quantitative RT-PCR(qRT-PCR)to explore its expression patterns in different tissues and in response to different abiotic stresses. Results showed that a TPS(trehalose-6-phosphate synthase)gene,MeTPS9,was cloned from cassava. This gene had a 2598-bp open reading frame,encoded 865 amino acids,and contained a TPS conserved domain. Phylogenetic analysis revealed that MeTPS9 had close genetic relationship with its homologs from Capsella grandiflora and Arabidopsis thaliana,and the sequence similarity was up to 76.2% and 75.6%,respectively. Gene structural variation demonstrated that a total of 66 mutation sites were identified between wild and cultivated cassava species,of which 11 were mis-sensed. Promoter assay showed that MeTPS9 contained drought-related motif MBS,cold-related motif LTR,heat-related motif HSE,and ABA-related motif ABRE. qRT-PCR analysis revealed that MeTPS9 expressed the highest in fibrous root but the lowest in leaf. In addition,the expression of MeTPS9 was significantly induced by drought,cold,and shade stress. MeTPS9 played a regulatory role in abiotic stress such as drought,cold,and shade treatment at the transcriptional level and it could be served as a candidate to further study its functions in abiotic stress defense in cassava.

Key words: cassava, MeTPS9, trehalose-6-phosphate synthase, structure variation, expression analysis, abiotic stress