利用SMART技术构建新吉细毛羊皮肤组织全长cDNA文库  

Constrction of a long-lenth cDNA library for Xinji Fine-wool Sheep skin tissue by use of SMART technique

作　　者：吴茜[1] 田可川[2] 刘武军[1] 张艳花[2] 吴伟伟[2] 徐新明[2] 张廷虎[2] 马晓燕[1] 黄锡霞[1] 

机构地区：[1]新疆农业大学动物科学学院,新疆乌鲁木齐830052 [2]新疆畜牧科学院,新疆乌鲁木齐830000

出　　处：《西北农林科技大学学报（自然科学版）》2010年第5期46-50,55,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：新疆自治区重大科技专项"细毛型;超细毛型细毛羊;绒山羊新品种(系)选育"(200731132-7);国家现代农业产业技术体系建设项目"绒毛用羊现代产业技术体系"(nycytx-40);国家科技支撑计划项目(2008BADB2B05)

摘　　要：【目的】探寻控制羊毛性状的相关基因,构建了新吉细毛羊皮肤组织全长cDNA文库。【方法】采用Clontech公司的CreaterTM SMARTTM cDNA library construction kit,用Biozol试剂提取新吉细毛羊皮肤组织总RNA后,以反转录酶SuperscriptTMⅡ反转录为第一链cDNA,然后通过LD-PCR合成并扩增ds cDNA,扩增产物经纯化、SfiⅠ酶切、过CHROMA SPIN-400柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB（带有SfiⅠA和B 2个位点）质粒载体中,最后用热击转化法将重组质粒转化到E.coliDH5α内,得到新吉细毛羊皮肤组织全长cDNA原始文库,扩增后的文库分装后用50 mL离心管保存。【结果】构建的cDNA文库约含4.2×105个独立克隆,重组效率为100%,插入片段多为0.5～3.0 kb,平均插入片段长度为1.3 kb,符合建库要求。【结论】经检测所构建的文库库容量满足基因筛选要求,可用于特异表达基因全长序列。【Objective】 The study was to construct a long-length cDNA library of skin tissue for Xinji Fine-wool Sheep to seek for genes related to wool trait.【Method】 The library was constructed by SMART technique using CreaterTM SMARTTM cDNA library construction kit.The total RNA was extracted from sheep skin tissue using Biozol isolation reagent.Single-strand cDNA was synthesized using SuperscriptTM Ⅱ reverse transcriptase,and double-strand cDNA was synthesized and amplified by long-distance PCR.The PCR products were purified by QIAquickTM PCR Purification kit.After digestion with SfiⅠand size fractionation using CHROMA SPIN-400 columns,ds cDNA was ligated to the SfiⅠ-digested,dephosphorylated pDNR-LIB vector.The ligation mixture was transformed into E.coli DH5α by thermal shock.【Result】 The cDNA library contained 4.2×105 independent clones with DNA inserts of 0.5-3.0 kb.The recombination rate was 100% and the average length of inserts was 1.3 kb.【conclusion】 The capacity of this library can meet the requirement for gene screening and provide a foundation to isolate specific genes in skin for Xinji Fine-wool Sheep.

关 键 词：新吉细毛羊 皮肤组织 CDNA文库 SMART技术 

分 类 号：S826.89[农业科学—畜牧学]