东亚钳蝎毒素BmKT四个同源基因的克隆及基因组织分析

Molecular Cloning and Genomic Organization of Four Homologues of Na^+-channel Toxins BmKT from Scorpion Buthus martensii Karsch

BmKT是从本室构建的cDNA文库中筛选到的1个α钠通道毒素,根据其全长cDNA序列设计引物,采用PCR法以蝎总基因组DNA为模板,获得4个BmKT的同源基因,分别命名为BmKT′和BmKTa、BmKTb、BmKTb′.序列分析表明:BmKT′和BmKTa基因含有大小分别为509bp和506bp的内含子,位于信号肽编码区内,插入信号肽-4位残基Gly密码子的第一个碱基G之后;而BmKTb和BmKTb′的内含子大小均为418bp.这4个BmKT的同源基因内含子符合GTAG拼接规律,其中BmKT′和BmKTa的内含子A+T含量分别为61.7%和61.9%,低于目前已报导的大多数蝎毒素基因A+T含量,大大低于它们第一外显子A+T含量(71.7%),略高于第二外显子A+T含量(55.5%);而BmKTb,BmKTb′的内含子A+T含量分别为75.8%和76.1%,与目前已报导的大多数蝎毒素基因A+T含量相似.BmKT′基因的外显子与BmKT基因cDNA所对应氨基酸序列仅在信号肽中-7位有一个氨基残基的差异(BmKT:Leu→BmKTa:Val);而BmKTa基因外显子所推断的氨基酸序列与BmKT前体比较,则在成熟肽的+54位发生了突变(BmKT:Lys→BmKTa:Asn),是与BmKT同源的一个新基因.BmKTb基因和BmKTb′基因所编码的前体肽与BmKT基因对应的前体肽同源性约为65%,显然BmKTb和BmKTb′是不同于BmKT的2个新基因(GenBank登录号:BmKT′,AY786186;BmKTa,AY676142;BmKTb,AY676140;BmKTb′,AY676141).

BmKT is a scorpion alpha-type toxin specially acting on Na^＋-channel, of which its full-length cDNA was isolated from the constructed cDNA library in our lab. Based on its cDNA sequence, the genomic DNA sequences encoding four homologues of BmKT cDNA, named BmKT＇, BmKTa, BmKTb, and BmKTb＇, individually, were obtained using the PCR method. Sequence analysis showed that the BmKT＇ contained a intron of 509 bp and BmKTa contained a intron of 506 bp inserted into the DNA region that encoding for the signal peptide. Similarly, BmKTb and BmKTb＇ contained a 418 bp intron, respectively, inserted into the same location as that of the BmKT＇ gene. The introns of these four genes matched the boundaries of the various introns with the consensus 5＇-GT/AG-3＇ site. The A ＋ T contents of BmKT＇ and BmKTa are relatively low （61.7% and 61.9% for BmKT＇ and BmKTa, respectively）; while those of BmKTb and BmKTb＇ are 75.8% and 76.1% respectively, similar to those of most scorpion toxin genes. The amino acid sequence deduced for BmKT＇ and the precursor of BmKT differed only at position - 7 （BmKT： Leu→BmKTa： Val） in the signal peptide; whereas that for BmKTa have a mutated position at ＋ 54 （ BmKT： Lys→BmKTa： Asn） in the mature peptide. The amino acid sequence deduced for BmKTb and BmKTb＇ share the 65% identification with the precursor of BmKT, which are apparently two newly-identified genes. （GenBank Accession Number： BmKT＇, AY786186; BmKTa, AY676142; BmKTb, AY676140; BmKTb＇, AY676141）