利用DSN和SMART^(TM)技术构建金龟子绿僵菌产孢时期均一化全长cDNA文库  

Construction of a Normalized Full-length cDNA Library of Metarhizium anisopliae var.acridum in the Sporulation Stage by DSN and SMART～(TM)

作　　者：张石柱[1] 王中康[1] 彭国雄[1] 曹月青[1] 殷幼平[1] 谢磊[1] 刘静[1] 夏玉先[1] 

机构地区：[1]重庆大学生物工程学院基因工程研究中心重庆市杀虫真菌农药工程技术研究中心功能基因及调控技术重庆市重点实验室,重庆400030

出　　处：《农业生物技术学报》2007年第5期884-887,共4页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.30170630);重庆市重点自然科学基金(No.8564)资助

摘　　要：为高效获得与绿僵菌产孢相关的全长cDNA,采用DSN(duplex-specific nuclease)均一化技术与SMARTTM(switching mechanism at 5′end of RNA transcript)建库技术相结合的方法,构建了杀虫真菌金龟子绿僵菌(Metarhizium anisopliae var.acridum)菌株CQMa102产孢时期的均一化全长cDNA文库。经检测,原始文库滴度为2.1×106cfu/mL,库容量超过6×106。随机挑取100个克隆,PCR方法测得文库重组率大于95%,插入片段平均长度1500bp。小规模测序分析表明,全长基因的比例超过60%。对组成性表达基因3-磷酸甘油醛脱氢酶G3PDH和微管蛋白β-tubulin均一化前后丰度检测表明,其丰度均有大幅度的下降,基本满足节约筛库的要求。In order to obtain full-length genes related to sporulation in Metarhizium anisopliae var. acridum CQMa102, a normalized cDNA library enriched in full-length sequences was constructed using DSN （duplex-specific nuclease）-normalization method combined with SMART^TM （switching mechanism at 5′end of RNA transcript） technique. The titer of un-amplified cDNA library was about 2.1×10^6 cfu/mL and contained 6×10^6 independent clones. The average cDNA inserts size was 1 500 bp with a recombination rate of 95%. Sequencing results and bioinformatics analysis of random picked clones showed that the ratio of full-length cDNA was about 60%. The abundance of transcripts G3PDH and β-tubulin decreased obviously compared with non-normalized samples. These results indicated that the normalized full-length cDNA library has been constructed successfully, which is convenient for further studyins the sporulation mechanism in Metarhizium.

关 键 词：绿僵菌 产孢基因 全长文库 均一化技术 switching mechanism at 5'end of RNA transcrip(tSMART^TM) duplex-spe-cific nuclease(DSN) 

分 类 号：S188[农业科学—农业基础科学]