【作者】 杨银凤； 郝永峰； 都格尔斯仁；

【Author】 Yang Yin-feng Hao Yong-feng Du Ge-er si ren (Laboratory of Animal Embryo and developmental biology, Inner Mongolia Agriculture University Huhhot 010018)

【机构】 内蒙古农业大学动物胚胎与发育生物学研究室；

【摘要】 为了获得驯鹿β-防御素reBD-1全长cDNA序列,根据已获得的reBD-1 cDNA的已知序列设计一条序列特异性引物作为上游引物,反转录引物中的部分序列即3’接合器引物作为下游引物,克隆reBD-1 cDNA的3’末端序列。另外,采用反向嵌套PCR RACE法,根据reBD-1 cDNA的已知序列,设计一条5’未端磷酸化的特异性反转录引物和两对特异性反向嵌套PCR引物,首先进行反转录(RT),然后将mRNA反转录成的cDNA 进行环化,最后进行反向嵌套巢式PCR,克隆reBD-1 cDNA的5’末端序列。结果成功的克隆出了reBD-1 cDNA 的3’和5’未端序列,从而得到372 bp的reBD-1 cDNA全序列,其中包含44bp 5’非翻译区(UTR)、192bp 的开放读码框(ORF)、终止密码子TAA、118bp的3’UTR和poly(A)15。reBD-1 cDNA全序列的获得为进一步研究其基因结构、基因表达和基因功能奠定了基础。更多还原

【Abstract】 In order to obtain the full-length cDNA, we cloned the 3’cDNA end of reindeer β-defensin (reBD-1) using anchored PCR RACE with a sequence specific primer designed basing on the known reBD-1cDNA sequence as upper primer and 3’sites adaptor primer as down primer. In addition, the 5’cDNA end was amplified using inverse nestd PCR with a specific RT primer whose 5’end is phosphated and two pairs specific inverse nested PCR primers, which are designed basing on the known reBD-1 cDNA sequence. The reBD-1 cDNA which come from reBD-1 mRNA by RT was encircled, then inverse nestd PCR is performed. The results indicate 3’and 5’cDNA end were amplified successfully. The full-length reBD-1 cDNA is 372bp and includes an 5’untranslated region (UTR) of 44 bp, a open reading frame(ORF)of 192bp, a stop codon of TAA, 3’ UTR and ploy(A)15. The obtaining of full-length reBD-1 cDNA is the essential foundation for the research on gene structures, gene expressions and gene functions.更多还原