蒙古沙冬青冷冻胁迫SMART cDNA文库的构建及序列分析
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国家自然科学基金项目,内蒙古科技计划项目


Construction of SMART cDNA library of Ammopiptanthus mongolicus under cold and freezing stresses and its sequence analysis
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    摘要:

    以强抗逆植物蒙古沙冬青 (Ammopiptanthus mongolicus)为材料,用SMART技术构建了其在冷冻胁迫下的全长cDNA文库。原始文库滴度为9.44?06 pfu.mL-1,重组率为98.3%,插入片段长度在0.5~2.5kb,平均达到1kb。扩增文库滴度为1.98?010pfu.mL-1。从原始文库中随机挑取480个阳性克隆进行序列分析,获得348个Unigene,其中有些是逆境应答相关基因和新基因。此外,以扩增文库为模板,经PCR方法克隆到读码框为1083bp的沙冬青类RD22基因。这些结果表明所建文库质量较高,可以满足后续研究的要求。

    Abstract:

    A cold and freezing-induced full-length cDNA library of Ammopiptanthus mongolicus, a plant with remarkable resistance to severe environments, was constructed by using SMART (Switching mechanism at 5’-end of RNA transcript)cDNA library construction method. The titer of the unamplified library is 9.44?06 pfu/mL; the recombination percentage of the library is 98.3%; the sizes of the insert cDNA fragments ligated to λTriplEx2 vector ranges from 0.5to 2.5b and averagely reaches up to 1.0kb. The titer of the amplified library is 1.98?010 pfu.mL-1. Four hundred and eighty positive clones selected from the unamplified library were sequenced and some stress responsive related genes and novel genes were found. Moreover, using the amplified library as template, a RD22-like gene with the complete coding region of 1083 base-pair was obtained by PCR method. All of the results indicated that the library quality is high and can satisfy the demands of follow-up researches.

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刘佳杰,林清芳,李连国,等.蒙古沙冬青冷冻胁迫SMART cDNA文库的构建及序列分析[J].植物遗传资源学报,2011,12(5):770-774.

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  • 收稿日期:2010-12-10
  • 最后修改日期:2011-03-21
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  • 在线发布日期: 2011-07-22
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