香樟1-脱氧-D-木酮糖-5-磷酸合酶基因的克隆与表达分析  

Cloning and Expression Analysis of DXS Gene in Cinnamomum camphora

作　　者：张瑶瑶 曹先爽 刘伟 王煜炜 王进 Zhang Yaoyao;Cao Xianshuang;Liu Wei;Wang Yuwei;Wang Jin(SFA Key Laboratory of Bamboo and Rattan Science and Technology,International Centre for Bamboo and Rattan,Beijing,100102)

机构地区：[1]国际竹藤中心,国家林业局竹藤科学与技术重点实验室,北京100102

出　　处：《基因组学与应用生物学》2020年第8期3570-3577,共8页Genomics and Applied Biology

基　　金：国家林业局948项目(2014-4-33);“十二五”国家科技支撑计划项目(2012BAD23B03)共同资助。

摘　　要：本研究通过克隆香樟植物的1-脱氧-D-木酮糖-5-磷酸合酶(DXS)基因,进行生物信息学分析,为香樟DXS基因的功能及其表达调控提供基础。根据香樟转录组数据中注释为DXS的核苷酸拼接序列,设计特异性引物,进行香樟DXS基因的克隆,应用荧光定量PCR方法分析DXS基因在香樟根、茎和叶中的表达量。克隆获得香樟DXS基因,其c DNA序列长度为2333 bp,开放读码阅读框(ORF)为2187 bp,编码728个氨基酸,含有转酮醇酶等3个保守结构域。香樟DXS蛋白定位于叶绿体,是一个不含信号肽、无跨膜结构域的亲水性稳定蛋白,且蛋白二级结构主要为α-螺旋和无规则卷曲。通过氨基酸序列同源性分析发现,香樟DXS氨基酸序列与黄兰和鱼腥草的DXS氨基酸序列相似性较高,均为85%。分子进化树结果表明,香樟DXS蛋白与黄兰DXS蛋白亲缘关系较近。组织特异性表达分析结果显示,DXS在香樟叶与茎中的表达量高于根。成功克隆获得香樟DXS基因,研究结果为进一步探讨香樟DXS基因在萜类物质生物合成途径中的作用提供理论依据。This study was to clone the 1-deoxy-D-xylulose-5-phosphate synthase(DXS)gene in Cinnamomum camphora and performed bioinformatics analysis to provide a foundation for further study on the function and expression regulation of CcDXS.Specific PCR primers were designed based on the transcriptome data of C.camphora and the full-length c DNA of DXS was cloned by RT-PCR.The expression levels of CcDXS in the roots,stems and leaves of C.camphorac were analyzed by quantitative fluorescence PCR.The results showed that the full-length cDNA sequence was 2333 bp and its open reading code reading frame(ORF)was 2187 bp,encoding728 amino acids.CcDXS contained three conserved domains,such as transketolase.The CcDXS protein was predicted as a hydrophilic and stable protein,located in the chloroplast and without transmembrane domain and signal peptide.The main secondary structure of CcDXS wereα-helix and random coil.The amino acid sequence of CcDXS showed 85%similarity with that of Magnolia champaca and Houttuynia cordata.Phylogenetic analysis revealed that CcDXS protein is more closely related to the DXS proteins from Magnolia champaca.The results of real-time quantitative PCR showed that CcDXS expression was higher in leaves and stems compared to the roots of C.camphora.These results could provide a theoretical basis for further study on the role of DXS gene in the biosynthesis pathway of terpenoids in C.camphora.

关 键 词：香樟 1-脱氧-D-木酮糖-5-磷酸合成酶 基因克隆 

分 类 号：Q943.2[生物学—植物学] S792.23[农业科学—林木遗传育种]