摘要
为了获得梅花鹿β-防御素-1(sika deerβ-defensin-1,siBD-1)cDNA全序列,本试验以梅花鹿舌黏膜组织内提取的总RNA为模板,根据前期已获得的siBD-1cDNA的已知部分序列设计引物,采用5′-RACE和3′-RACE技术分别扩增5′-和3′-末端序列,将此扩增产物克隆入pMD18-T载体,进行PCR、双酶切鉴定及序列测定与分析。结果表明,成功克隆出长度约为172和299bp的siBD-1cDNA 5′-和3′-末端序列,从而得到418bp的siBD-1cDNA全序列(GenBank登录号:HM588696.1),其中包含89bp 5′-非翻译区(UTR)、192bp的开放阅读框(ORF)、终止密码子TAA、118bp的3′-UTR和Poly(A)16。同源性比对结果显示,siBD-1cDNA与水牛的肠防御素(BEBD)同源性最高,为90.6%,与牛(EBD、LAP、TAP、BNBD-4)、山羊(GBD-1、GBD-2)、驯鹿(reBD-1)、绵羊(sBD-1、sBD-2)和骆驼(caBD-1)的防御素cDNA的同源性较高,分别为83.2%、83.1%、87.3%、87.0%、87.5%、87.5%、84.4%、79.9%、77.1%和70.5%;与马(hoBD-1)和猪(pBD-1)的同源性较低,为60.3%和72.4%;而与人(hBD-2)的同源性最低,为16.0%。siBD-1成熟肽由38个氨基酸残基组成,其中包含9个带正电荷的氨基酸残基。
In order to obtain the full-length cDNA of sikaβ-defensin-1(siBD-1),5′-RACE and 3′-RACE primers were designed according to the partial sequences of siBD-1that obtained from this study group,cDNA ends(RACE)technology were used to amplify siBD-1cDNA from the total RNA of tongue mucosa tissue in sika,and amplified products were cloned into pMD18-T vector and subjected to PCR,restriction endonuclease digestion and sequencing.The results indicated that the length of siBD-1cDNA 5′-and 3′-end were about 172 and 299bp.The full-length siBD-1cDNA was 418bp(GenBank accession No.HM588696.1)and includes an 5′-untranslated region(UTR)of 89 bp,a open reading frame(ORF)of 192 bp,a stop codon of TAA,3′-UTR of118 bp and Ploy(A)16.The sequence homology showed that siBD-1shared the greatest identity(90.6%)with buffalo entericβ-defensin,the higher identity with cattle(EBD,LAP,TAP,BNBD-4),goats(GBD-1,GBD-2),reindeer(reBD-1),sheep(sBD-1,sBD-2)and camel(caBD-1),that were 83.2%,83.1%,87.3%,87.0%,87.5%,87.5%,84.4%,79.9%,77.1% and70.5%,respectively,and correspondingly low identity were 60.3%and 72.4% with horse(hoBD-1),pig(pBD-1),the lowest was 16.0% with human(hBD-2).The mature peptide was consisted of 38 amino acids,and contained 9positively charged residues.
引文
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