王丰青, 周艳, 黄勇, 李明杰, 田云鹤, 冯法节, 陈新建, 张重义. 地黄扩展蛋白基因RgEXPA10的克隆与表达分析J. 药学学报, 2015,50(2): 233-240.
引用本文: 王丰青, 周艳, 黄勇, 李明杰, 田云鹤, 冯法节, 陈新建, 张重义. 地黄扩展蛋白基因RgEXPA10的克隆与表达分析J. 药学学报, 2015,50(2): 233-240.
WANG Feng-qing, ZHOU Yan, HUANG Yong, LI Ming-jie, TIAN Yun-he, FENG Fa-jie, CHEN Xin-jian, ZHANG Zhong-yi. Cloning and expression analysis of the expansin gene RgEXPA10 in Rehmannia glutinosaJ. Acta Pharmaceutica Sinica, 2015,50(2): 233-240.
Citation: WANG Feng-qing, ZHOU Yan, HUANG Yong, LI Ming-jie, TIAN Yun-he, FENG Fa-jie, CHEN Xin-jian, ZHANG Zhong-yi. Cloning and expression analysis of the expansin gene RgEXPA10 in Rehmannia glutinosaJ. Acta Pharmaceutica Sinica, 2015,50(2): 233-240.

地黄扩展蛋白基因RgEXPA10的克隆与表达分析

Cloning and expression analysis of the expansin gene RgEXPA10 in Rehmannia glutinosa

  • 摘要: 以地黄(Rehmannia glutinosa)叶片cDNA为模板, 克隆地黄扩展蛋白基因, 获得一条972 bp的cDNA序列, 包含了762 bp的开放阅读框(ORF), 命名为RgEXPA10, GenBank登录号为KF011918, 编码253个氨基 酸。以基因组DNA为模板, 扩增出一条1 207 bp的序列, 测序结果显示包含3个外显子和2个内含子, GenBank登录号为KF011919。应用生物信息学方法分析其氨基酸序列结构特征, 结果显示, RgEXPA10包含DPBB_1和Pollen_allerg_1结构域, 有26氨基酸的核定位序列和19氨基酸的跨膜结构域。进化分析发现在拟南芥26个α扩展蛋白中, RgEXPA10与AtEXPA8的同源性最高。在22个植物的同源蛋白中, 地黄RgEXPA10与番茄扩展蛋白的亲缘关系最近。以实时荧光定量PCR(qRT-PCR)检测RgEXPA10基因的表达量, 发现RgEXPA10在地黄未展开的幼嫩叶片里表达最强, 其次为膨大中的块根, 在衰老的叶片中表达最弱。出苗后60和90天时, 在地黄块根中的表达较强。在连作、盐和渍水3种逆境胁迫下, 地黄叶片内RgEXPA10的表达量均显著降低。为研究RgEXPA10在地黄发育和逆境胁迫中的分子功能奠定基础。

     

    Abstract: Using cDNA from Rehmannia glutinosa leaf as template, a 972 bp fragment of expansin gene which containing a 762 bp ORF that encoded 253 amino acids, was cloned, named RgEXPA10, which GenBank accession number for this gene is KF011918. A 1 207 bp genomic sequence of RgEXPA10 was amplified by PCR with leaf DNA as template, sequencing analysis revealed that three exons and two introns in RgEXPA10 genomic sequence, and which GenBank accession number is KF011919. Molecular and bioinformatic analyses indicated that RgEXPA10 protein have DPBB_1 and Pollen_allerg_1 domain, also including a 26 aa nuclear localization signal and a 19 aa transmembrane region. Phylogenetic analysis revealed that RgEXPA10 showed the highest homology with AtEXPA8 among the 26 α-expansins in Arabidopsis thaliana. However, the RgEXPA10 indicated the highest homology with the expansin from Solanum lycopersicum among 22 plant species. Expression patterns using qRT-PCR analysis showed that RgEXPA10 mainly expressed in unfolded leaf, followed by the tuberous root at stage of expanding period, and rarely expressed in senescing leaf. And RgEXPA10 showed higher expression level in tuberous root at 60 and 90 days after emergence. The transcription level of RgEXPA10 significantly reduced under all the three stresses including continuous cropping conditions, salinity and waterlogging. This study will lay foundations for molecular function in development and regulation of different stresses for R. glutinosa.

     

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