东亚钳蝎毒素BmKT四个同源基因的克隆及基因组织分析

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509 bp和506 bp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418 bp．这4个BmKT的同源基因内含子符合GT/AG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）

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

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 ofBmKTb 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: LysBmKTa: 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)