马铃薯腐烂茎线虫L 型半胱氨酸蛋白酶新基因(Dd-cpl-1) 的克隆与序列分析

Cloning and sequence analysis of a new cathepsin L-like cysteine proteinase gene from Ditylenchus destructor

摘要:

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 个内含子，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Asn³⁴³) 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。

Abstract:

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 cathepsin 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.