RACE法在连翘花蕾全长cDNA文库构建中的应用  

Application of RACE method on the construction of full-length cDNA library for floral buds in Forsythia suspense

作　　者：刘伟[1,2] 孙海峰[1,2] 郭小青[2] 张丽增[2] 秦雪梅[1,2] 

机构地区：[1]山西大学化学化工学院,太原030006 [2]山西大学中医药现代研究中心

出　　处：《山西医科大学学报》2009年第10期902-905,共4页Journal of Shanxi Medical University

基　　金：山西省科技基础条件平台建设基金资助项目(2005091016-0502)

摘　　要：目的建立新的连翘花蕾全长cDNA文库构建方法。方法应用GeneRacerTM试剂盒,采用改良RNA连接酶介导的快速扩增cDNA末端(RNA ligase-mediated rapid amplication of cDNA ends,RLM-RACE)技术构建连翘花蕾全长的cDNA文库,通过计算菌落数、蓝白斑数、酶切鉴定、序列分析等手段评价文库质量。结果通过增加模板用量的方法成功构建了连翘花蕾全长cDNA文库,该文库容量为3.47×105,重组率为81.6%,插入片段多集中在500-1000bp之间,所占比例为71%;随机测序的cDNA克隆中均含有完整开放阅读框的全长cDNA序列。结论应用RACE法成功构建了连翘花蕾全长cDNA文库。Objective To establish a new method for the construction of full-length cDNA library of floral buds from Forsythia suspense （Thunb.） Vahl. Methods The cDNA was prepared using GeneRacerTM kit with some improvement. Colony forming unit （ CFU）, numbers of blue and white clones, enzyme digestion, and sequence analysis were used to evaluate the quality of constructed cDNA library. Results The library capacity was 3.47 × 10^5 ,the recombination rate was 81.6% ,and about 71% of inserts was 500 bp to 1 000 bp in length. And all randomly sequenced inserts were full-length cDNA clones containing intact open reading frames. Conclusion The cDNA library is successfully constructed with GeneRacerTM kit, which is very useful for further study on the molecular mechanism of floral development and secondary metabolism in Forsythia suspense （ Thunb. ） Vahl. And the new method can be used for the construction of full-length cDNA library in other species.

关 键 词：连翘 全长CDNA文库 构建 RACE 

分 类 号：R284[医药卫生—中药学]