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Plant Diversity ›› 2009, Vol. 31 ›› Issue (02): 146-152.DOI: 10.3724 SP.J.1143.2009.08181

• 研究论文 • 上一篇    下一篇

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

尹红菊, 王留阳, 高 茜, 高大海, 张东远, 刘建全   

  1. 兰州大学干旱与草地生态教育部重点实验室分子生态研究所, 甘肃兰州 730000
  • 收稿日期:2008-09-24 出版日期:2009-04-25 发布日期:2009-04-25
  • 通讯作者: 尹红菊

cDNA Library Construction and Expressed Sequence Tags (ESTs) Analyses of an Alpine Plant Species Gentiana officinalis (Gentianaceae)

YIN Hong-Ju , WANG Liu-Yang , GAO Qian , GAO Da-Hai, ZHANG Dong-Yuan, LIU Jian-Quan   

  1. Institute of Molecular Ecology, Key Laboratory of Arid and Grassland Ecology , School of Life Sciences, Lanzhou University , Lanzhou 730000, China
  • Received:2008-09-24 Online:2009-04-25 Published:2009-04-25
  • Contact: YIN Hong-Ju

摘要: 黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。

关键词: 黄管秦艽, 基因组学, cDNA 文库, 表达序列标签, 龙胆属

Abstract: Gentiana officinalis, an alpine plant , one of the widely used Tibetan traditional medicines . In this study, total RNA was extracted from the whole plant of flowering individuals of this species , and a cDNA expression library was constructed using CreatorTM SMARTTM cDNA Library Construction Kit . The results showed that the titer of the cDNA expression library was 1. 2×107 pfu􊄯ml and the efficiency of recombination was 95 . 9% . The average length of insert fragments in the library was longer than 500 bp . A total of 181 valid ESTs were obtained from random sequencing of 343 clones. Further
bioinformatic analyses suggested they represented 144 unique clonal sequences in which 55 sequences showed high homology to previously identified genes in Gentianaceae or other plants, 35 sequences matched to other uncharacterized expressed sequence tags (ESTs) , and 54 sequences showed no well matches to available sequences in DNA databases . No protein matched to the latter two sorts of ESTs ( 89) . Fifty-five ESTs with matched proteins were involved in a series of diverse functions: protein expression (35% ) , photosynthesis (22% ), metabolism ( 18%), defense (11%), membrane transport (5%), cell division (5% ) , chromosome metabolism ( 2% ) and signaling components (2%). At last , RT-PCR primers
were designed according to the effective ESTs to amplified the cDNAs of G. officinalis, which further verified the accuracy of the ESTs . This cDNA library provided a critical basis for further analyses of functional genes and gene expression in this alpine species. In addition, these ESTs could be used to design functional nuclear primers for studying population genetics of this species and closely related species .

Key words: Gentiana officinalis