蒙古沙冬青冷冻胁迫SMART cDNA文库的构建及序列分析

Construction of SMART cDNA Library of Ammopiptanthus mongolicus under Cold and Freezing Stresses and Its Sequence Analysis

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cD-NA文库。原始文库滴度为9.44×106pfu/ml,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1.98×1010pfu/ml。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。结果表明所建文库质量较高,可以满足后续研究的要求。

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 × 10^6 pfu/ml; the recombination percentage of the library is 98.3% ; the sizes of the insert cDNA fragments ligated to λTriplEx2 vec- tor ranges from 0.5kb to 2.5kb and averagely reaches up to 1.0kb. The titer of the amplified library is 1.98 ×10^10 pfu/ml. 480 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 RD22-1ike 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 it can satisfy the demands of follow-up researches.