马铃薯腐烂茎线虫2-Cys型过氧化物还原酶基因的克隆及低剂量杀线虫剂对其表达的影响  

Cloning and Sequence Analysis of a 2-cysteine Peroxiredoxin Gene from Ditylenchus destructor and the Effects of Low-dose Nematicides on Its Expression

作　　者：何旭峰[1,2] 彭焕[2] 彭德良[2] 黄文坤[2] 贺文婷[2] 丁中[1] 

机构地区：[1]湖南农业大学生物安全科学技术学院/植物病虫害生物学与防控湖南重点实验室,长沙410128 [2]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100193

出　　处：《农业生物技术学报》2013年第6期698-706,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31071667)

摘　　要：过氧化物还原酶(Peroxiredoxins,Prx)属抗氧化蛋白超家族,其催化活性依赖于半胱氨酸的存在。在该家族成员中,2-cysteine型过氧化物还原酶(2-Cys Prx)在抵抗外源性ROS保护植物线虫表皮免受氧化性损害以及线虫生长发育方面具有重要作用。为了解低剂量杀线虫剂对2-Cys Prx基因表达的影响,本研究采用RT-PCR和RACE技术克隆了马铃薯腐烂茎线虫(Ditylenchus destructor)2-Cys Prx基因,并利用Real-time PCR检测了马铃薯腐烂茎线虫经低剂量(10、1、0.1、0.01、0.001和0.0001μg/mL)杀线虫剂甲氨基阿维菌素苯甲酸盐和涕灭威处理24 h后,2-Cys Prx基因表达量的变化。结果表明,该基因cDNA全长为784 bp,编码196个氨基酸,命名为Dd-Prx-1(GenBank登录号:JQ609353)。同源性分析结果表明,Dd-Prx-1与其他线虫的2-Cys Prx具有较高的同源性。Dd-Prx-1的基因组由6个外显子和5个内含子组成。蛋白质预测N端没有明显的信号肽,但有一个含有23个亲水氨基酸的跨膜结构域。qRT-PCR分析结果表明,经低剂量甲氨基阿维菌素苯甲酸盐和涕灭威处理后,马铃薯腐烂茎线虫Dd-Prx-1基因的表达量与对照相比较无显著性差异(P<0.05)。结果为深入研究植物线虫应对低剂量杀线虫剂的机制提供了基础资料。Peroxiredoxins（Prxs）are a superfamily of cysteine-dependent peroxidases that decompose hydrogen peroxide. Among the family, typical 2-cysteine Peroxiredoxins （2-Cys Prxs） are multifunctional proteins. In plant nematode, 2-Cys Prxs play an important role in nematode development in planta or incountering radicals produced by the plant as a defense response to parasitism. In order to understand the effect of low dose of nematicides on 2- Cys Prxs mRNA expression, a novel 2- Cys Prx gene was cloned froml）itylenchus destructor using RT-PCR and RACE techniques, and its mRNA expression of D. destructor treated by low dose（10, 1,0.1,0.01,0.001 and 0.0001 μg/mL） nematicides of emanectin benzoate and aldicarb weremonitored using Real-time PCR after 24 h. The results showed that the full-length cDNA was 784 bp with an open reading frame （ORF） of 591 bp encoding 196 amino acids residues, named Dd-prx-1（GenBank accessionNo. JQ609353）. The protein had a predicted molecular weight of 21 850.09 D and a hypothetical pl of 6.83. The structure of Dd-Prx-1 genomic DNA include 6 extons and 5 introns. The N-terminal of predicted proteinhad no apparent signal peptide, but there was one transmembrane domain containing of 23 hydrophilic amino acids. Homology analysis showed that the deduced Dd-PRX- 1 protein was highly homologous to the 2-cysteine peroxiredoxin in other nematodes. After treated by low dose emanectin benzoate and aldicarb, the expression levels of Dd-Prx-1 had no significant difference compared with control （P〈0.05）. The resultsprovide the useful information for further research mechanisms of nematodes response to low dose of nematicides.

关 键 词：马铃薯腐烂茎线虫 过氧化物还原酶 杀线虫剂 基因表达 

分 类 号：S432.45[农业科学—植物病理学]