毛冠鹿大脑组织全长cDNA文库构建  

Construction of the cDNA Library from Cerebrum of Tufted Deer

作　　者：汤文文[1] 张文[1] 曹祥荣[1] 张锡然[1] 徐春茂 王强[3] 胡均 

机构地区：[1]南京师范大学生命科学学院,南京210097 [2]安徽省皖南野生动物救护中心,休宁245400 [3]四川省成都动物园,成都610081

出　　处：《动物学杂志》2006年第1期53-59,共7页Chinese Journal of Zoology

基　　金：国家自然科学基金(No.30370789);江苏省教委自然科学基金(No.02KJD180006)

摘　　要：运用SMART技术构建了毛冠鹿(Elaphodus cephalophus)大脑组织全长cDNA文库。提取大脑组织总RNA,Oligotex mRNA Kit纯化、获得poly(A)+RNA,以CDSⅢ/3′PCR引物进行逆转录,LD-PCR扩增获得全长双链cDNA,经SfiⅠ酶切及柱层析分离后,500 bp以上的片段与载体λTripIEx2连接,体外包装得到cDNA文库。经鉴定原始文库滴度为5.1×105pfu/ml,扩增后文库滴度为1.5×109pfu/ml,重组率达到85%以上,插入片断平均长度约为1.0 kb,说明构建文库质量符合要求,可用于大脑特异表达基因的筛选。从该文库中克隆到了rig基因全长,包含5′和3′非编码区,从第43至477个核苷酸为一完整阅读框(ORF),此阅读框可编码一个145氨基酸的rig蛋白。The cerebrum cDNA library of Tufted Deer （ Elaphodus cephalophus ） was constructed using SMART technique.Total RNA was extracted from cerebrum tissue of Tufted Deer,and poly （A）^＋ RNA was purified further by Oligotex mRNA Kit. CDS Ⅲ/ 3＇ PCR Primer was used to prime the reverse transcription. The double-strand cDNA was amplified by LD-PCR, and digested by Sfi Ⅰ. After cDNA size fraction by CHROMA SPIN-400, fragments over 500 bp were collected and ligated with λTripIEx2 vector. After packaging in vitro, the full-long cDNA library of cerebrum tissue was constructed successfully. The library contains 5.1 × 10^5 pfu/ml clones and the amplified library has a titer of 1.5 × 10^9 pfu/ml,in which 85% clones are recombinant and the average sizes of inserted cDNAs are about 1.0 kb. These results show that the cerebrum cDNA library is qualified for screening genes. A full-length rig gene with 5＇ and 3＇untranslated regions was isolated from the cDNA library. Sequence analysis shows that this 541 bp cDNA spans an open reading frame from nucleotide 43 to 477,encoding a 51-aminoacid rig protein.

关 键 词：毛冠鹿 大脑 CDNA文库 SMART技术 

分 类 号：Q785[生物学—分子生物学]