猕猴桃溃疡病菌漆酶基因的克隆与功能分析

Cloning and analysis of the expression of laccase genes in Pseudomonas syringae pv. actinidae

为探明猕猴桃溃疡病病菌漆酶基因的序列特征与功能,研究从12株溃疡病病菌丁香假单孢杆菌猕猴桃致病变种(Pseudomonas syringae pv.actinidae)中扩增获得漆酶基因,分别命名为Psalac JF、Psalac HY、Psalac GC、Psalac HWD、Psalac INS、Psalac7285、Psalac LT12、Psalac LT16、Psalac LT26、Psalac349、Psalac Kw30和Psalac K3。序列分析结果显示上述序列之间只有少许核酸位点有差异,基因编码全长都为1374 bp,BLAST预测基因编码有457个氨基酸序列(包括N-端20个氨基酸的信号肽),启动子位于基因上游175 bp。生物信息学分析发现该基因具有漆酶特有的4个Cu2+活性位点,且活性位点高度保守。系统进化分析结果显示该基因编码序列与多种细菌的漆酶基因同源关系很近,其中与P.fluorescens Pf0-1的同源性最高。研究结果表明溃疡病病菌中普遍含有具备细菌漆酶基因家族的序列特性,推测该基因可能参与调控溃疡病病菌致病性和Cu2+耐受性。

The objective of this study was to reveal sequence features of laccase,which would be used in further study of pathogenesis of kiwifruit bacterial canker caused by Pseudomonas syringae pv. actinidae. 12 laccase genes homologs Psalac JF,Psalac HY,Psalac GC,Psalac HWD,Psalac INS,Psalac7285,Psalac LT12,Psalac LT16,Psalac LT26,Psalac349,Psalac Kw30 and Psalac K3 were cloned from different Pseudomonas syringae pv. actinidae strains isolated from diverse regions and countries. The results showed that the complete DNA sequence of laccase gene were 1374 bp in length,encoding a putative proteins of 457-amino acid. All canker strains share the same amino acid sequences and small nucleic acid sequence differences,containing four highly conservative Cu2 ＋active sites. Promoter is located in the upstream gene with 175 bp. Phylogenetic analysis indicated that putative laccase sequence was homologous to P. fluorescens Pf0-1 laccase CP000094. 2. The studies suggested that laccase gene had the sequence characteristics of laccase gene family in bacteria,which meant that laccase might involve in the pathogenicity and copper-resistant of Pseudomonas syringae pv.actinidae.