L型半胱氨酸蛋白酶基因(Cathepsin L-like cysteine proteinase gene)为与植物寄生线虫寄生能力相关的多功能基因.运用RT-PCR和RACE的方法从马铃薯腐烂茎线虫Ditylenchus destructor中克隆出1个L型半胱氨酸蛋白酶新基因Dd-cpl-1(GenBank登录号为GQ180107).该基因Dd-cpl-1 cDNA全长序列含有1个1 131 bp的开放性阅读框(ORF),编码376个氨基酸残基,其5'末端及3'末端分别含有29 bp和159 bp的非编码区(UTR).Dd-cpl-1内含子外显子结构分析结果表明,其基因组序列包含7个内舍子,且各内含子两端剪接位点序列遵守GT/AG规则.Dd-cpl-1基因推定的蛋白Dd-CPL-1与松材线虫L型半胱氨酸蛋白酶高度同源,一致性达到77%.以不同物种中L型半胱氨酸蛋白酶氨基酸序列进行比对分析,推测推定的蛋白Dd-CPL-1含有L型半胱氨酸蛋白酶基因家族高度保守的催化三联体(Cys183,His322和Ash343)以及ERFNIN基系和GNFD基系.半胱氨酸蛋白酶系统发育分析表明,Dd-cpl-1属于由L型半胱氨酸蛋白酶组成的进化分支.Dd-cpl-1的这些序列特征进一步表明其为L型半胱氨酸蛋白酶基因.这是首次在马铃薯腐烂茎线虫中克隆到的L型半胱氨酸蛋白酶,为今后在蛋白水平对其进行进一步的功能分析提供基础.%The Cathepsin L-like cysteine proteinase genes (cpls) are multifunction genes related to the parasitic abilities of plant parasitic nematodes.A new cathepsin L-like cysteine proteinase gene (Dd-cpl-1) (GenBank Accession GQ 180107) was cloned from Ditylenchus destructor by RT-PCR and RACE.The cDNA sequence consisted of a 1 131 bp open reading frame (ORF) encoding 376 amino acid residues that were franked by a 29 bp 5'-untranslated region (UTR) and a 159 bp 3'-UTR.Genomic sequence analysis showed that Dd-cpl-1 contained 7 introns, obeyed the GT/AG rule in the splice-site junctions.Homology analysis showed that the identity was 77％ between Dd-cpl-1 deduced protein Dd-CPL-1 and eathepsin L-like cysteine proteinase of Bursaphelenchus xylophilus.Multi-sequence alignment indicated that there were the catalytic triad (Cys183, His322 and Asn343) and two motifs ERFNIN motif and GNFD motif in deduced protein Dd-CPL-1.Cysteine proteinases phylogenetic analysis showed that Dd-cpl-1 belonged to the sub-clade of cathepsin L-like cysteine proteinases.
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