摘要
利用SMART(switching mechanismat 5′end of RNA transcript)技术,采用Clontech公司的SMARTTMcDNALi-brary Construction Kit构建扩展莫尼茨绦虫成虫全长cDNA文库。用Trizol Reagent提取总RNA,用Oligotex mRNA Kits分离mRNA,以逆转录酶PowerScriptTM合成第一链cDNA,通过LD-PCR合成并扩增双链cDNA;扩增产物经蛋白酶K消化、SfiⅠ酶切、过SPIN-400TM柱去除小片段,连接到SfiⅠ消化过的pBluescriptⅡSK*质粒载体中,最后将重组质粒转化到E.coliDH5α内得到原始文库,扩增后,将文库保存于96孔细胞培养板中。经测定,构建的cDNA文库滴度为2.52×105PFU/mL;重组率为94.7%,插入片段长度多在0.5～3kb之间,平均长度约1.2kb。经5′端测序获得2642条有效序列,归并为1081条unigene。结果表明成功构建了扩展莫尼茨绦虫成虫全长cDNA文库,获得了较丰富的扩展莫尼茨绦虫成虫表达基因,为筛选扩展莫尼茨绦虫的功能基因全长序列奠定了基础。
A cDNA library for Moniezia expansa was constructed by switching mechanism at 5＇ end of RNA transcript （SMART） technique using SMARTTM cDNA Library Construction Kit （Clontech）. The total RNA was extracted from M. expansa using Trizol Reagent and mRNA was purified using Oligotex mRNA Kits. Single-strand eDNA was synthesized using PowerSeript^TM reverse transcriptase, and double-strand cDNA was synthesized and amplified by long-distance PCR. The PCR products were digested by proteinase K. After digestion with Sfi Ⅰ and size fraetionation using SPIN-400^TM columns, SMART eDNA was ligated to pBluescript Ⅱ SK^＊ vector. The ligation mixture was transformed into E. coli DH5a. The recombination rate of the library was 94.7%, and the titre was 2.52X105 PFU/mL. The size of inserts was about 0.5 to 3 kb, and the average length was 1.2 kb. 2642 ESTs from 5＇-ends of the eDNA clones representing 1081 unigenes were obtained. The result dis- plays M. expansa eDNA library is successfully constructed, and could serve as a valuable resource for screening and isolating specific genes for M. expansa.
出处
《中国畜牧兽医》
CAS
北大核心
2009年第7期51-55,共5页
China Animal Husbandry & Veterinary Medicine
基金
国家科技部973前期研究专项(2006CB708512)
