东北梅花鹿鹿茸尖端组织全长cDNA文库的构建  

Construction of Full-length cDNA Library for Antler Tip Tissue of Sika Deer

作　　者：郝丽[1] 李和平[1] 严厉[1] 

机构地区：[1]东北林业大学,哈尔滨150040

出　　处：《东北林业大学学报》2009年第11期120-122,共3页Journal of Northeast Forestry University

基　　金：黑龙江省自然科学基金(C200725)资助项目

摘　　要：为克隆出与鹿茸生长发育相关基因的全长序列,采用SMART技术构建了东北梅花鹿鹿茸尖端组织的全长cDNA文库。用SVTotal RNAIsolation System试剂盒提取总RNA,以逆转录酶PowerScriptTM反转录合成第一链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CHROMASPIN-400柱去除小片段后,连接到SfiⅠ消化过的pDNR-LIB质粒载体中,最后用电转化法将重组质粒转化到E.coliDH5α内得到原始文库。经测定,构建的原始文库约含有2.56×106个重组子,插入片段多在0.5～2 kb之间,平均插入片段长度约1.1 kb,重组效率接近100%。结果表明,东北梅花鹿鹿茸尖端组织的全长cDNA文库已构建成功。A study was conducted to construct full-length eDNA library from antler tip tissues of Sika Deer （ Cervus nippon hortulorum） by SMART technique in order to clone new special genes for development of antler. The total RNA was extracted using SV Total RNA Isolation System. Single-strandod eDNA was synthesized using PowerScrlptTM reverse transcriptase, and double-stranded eDNA was synthesized and amplified by long-distance PCR. Tile PCR products were digested by proteinase K and purified. After digestion with Sfi I and size fraetionation using CHROMA SPIN -400TM Columns, SMART eDNA was ligatod to the Sfi I-digested, dephosphorylatod pDNR-LIB vector, and the ligation mixture was transformed into E. coli DH5α by eleetroporation. The primary eDNA library contained 2.56 ×10^6independent clones with DNA inserts of 0.5 - 2.0 kb, the average size of inserted cDNAs was 1.1 kb, and the recombination percentage was about 100%. Results showed that the full-length eDNA library from antler tip tissues of Sika Deer was successfully constructed.

关 键 词：东北梅花鹿 鹿茸 全长CDNA文库 SMART技术 

分 类 号：Q78[生物学—分子生物学]