SMART技术构建栀子cDNA文库

目的:构建栀子叶片cDNA文库。方法:提取栀子叶片总RNA。利用SMART技术合成双链cDNA。双链cDNA经限制酶Sfil酶切后与pDNR-LIB质粒连接。利用电刺激转化法将重组质粒导入E.coli DH5α而获得文库。利用PCR法检测文库的重组率。结果:原始文库滴度为2.63×105cfu/ml。随机检测文库中的15个克隆,表明重组率约为86.7%。选择14个插入片段的长度在400bp以上的克隆进行测序和生物信息学分析,结果预测的全长基因占所检测序列的64.3%。结论:成功构建了栀子叶片的cDNA文库,为栀子基因的结构和功能的研究提供了基础。

Construction a cDNA Library of Gardenia jasminoides Ellis with SMART Technique

Obiective:To construct a cDNA Library of Gardenia jasminoides Ellis.Method:Total RNA was extracted from the leaves of Gardenia jasminoides Ellis.The SMART technique was used to synthesize double-strand cDNA.After digested by Sfil restriction enzyme,the cDNA was ligated to pDNR-LIB vector.The plasmids were transformed into E.coli DH5α by electroporation.The quality of the cDNA library was tested by PCR.Result:The titer of the cDNA library is estimated to be 2.63×10~5 cfu/ml.The recombinant percentage of the library was determined as 86.7% by random screening fifteen clones.Fourteen inserts that longer than 400bp were sequenced and analyzed,the possible full-length cDNA sequences account for 64.3%.Conclusion:A cDNA Library from the leaf of Gardenia jasminoides Ellis was constructed;it provides an effective tool to obtain the gene information of Gardenia jasminoides Ellis.