用SMART技术构建扩展莫尼茨绦虫成虫全长cDNA文库

›› 2009, Vol. 36 ›› Issue (7): 51-55.

用SMART技术构建扩展莫尼茨绦虫成虫全长cDNA文库

张慧^1,2，赵文娟^1,2，韩猛立^1,2，王新华²，薄新文²

(1.石河子大学动物科技学院，石河子 832003； 2.新疆兵团绵羊繁育生物技术重点实验室，石河子 832000)

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2009-07-20 发布日期:2009-07-20
通讯作者: 薄新文

Construction of cDNA Library from Moniezia expansa by SMART Technology

ZHANG Hui^1,2, ZHAO Wen-juan^1,2, HAN Meng-li^1,2, WANG Xin-hua², BO Xin-wen²

(1.College of Animal Science & Technology, Shihezi University, Shihezi 832003, China;2.Genetic Engineering Laboratory, the Breed & Biotechnology Key Laboratory of Sheep in Xinjiang Bingtuan, Shihezi 832000, China)

Received:1900-01-01 Revised:1900-01-01 Online:2009-07-20 Published:2009-07-20
Contact: BO Xin-wen

摘要/Abstract

摘要： 利用SMART(switching mechanism at 5′ end of RNA transcript)技术，采用Clontech公司的SMARTTM cDNA Library 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PowerScriptTM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、SfiⅠ酶切、过SPIN-400TM柱去除小片段，连接到SfiⅠ消化过的pBluescript Ⅱ SK*质粒载体中，最后将重组质粒转化到E.coli DH5α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2.52×105 PFU/mL；重组率为94.7%，插入片段长度多在0.5～3 kb之间，平均长度约1.2 kb。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。

关键词: 扩展莫尼茨绦虫; cDNA文库; SMART

Abstract: A cDNA library for Moniezia expansa was constructed by switching mechanism at 5′ end of RNA transcript (SMART) technique using SMARTTM 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 cDNA was synthesized using PowerScript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 fractionation using SPIN-400TM columns, SMART cDNA was ligated to pBluescript II SK* vector. The ligation mixture was transformed into E.coli DH5α. The recombination rate of the library was 94.7%, and the titre was 2.52×105 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 cDNA clones representing 1081 unigenes were obtained. The result displays M.expansa cDNA library is successfully constructed，and could serve as a valuable resource for screening and isolating specific genes for M.expansa.

Key words: Moniezia expansa; cDNA library; SMART

中图分类号:

Q785

张慧;赵文娟;韩猛立;王新华;薄新文. 用SMART技术构建扩展莫尼茨绦虫成虫全长cDNA文库[J]. , 2009, 36(7): 51-55.

ZHANG Hui;ZHAO Wen-juan;HAN Meng-li;WANG Xin-hua;BO Xin-wen. Construction of cDNA Library from Moniezia expansa by SMART Technology[J]. , 2009, 36(7): 51-55.

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