柑橘全爪螨谷氨酸脱羧酶基因PcGAD克隆与表达模式分析.docVIP

  柑橘全爪螨谷氨酸脱羧酶基因 PcGAD 克隆 与表达模式分析# 夏文凯，钟锐，丁天波，廖重宇，豆威，王进军** 5 10 15 （西南大学植物保护学院，昆虫学及害虫控制工程重庆市市级重点实验室，重庆 400716） 摘要：谷氨酸脱羧酶（glutamate decarboxylase, GAD）是广泛存在于昆虫体内的一种酶，能 催化谷氨酸脱羧形成γ-氨基丁酸(γ-aminobutyric acid, GABA)。 GAD 在生物合成中，是催 化谷氨酸脱羧行成 GABA 的唯一酶，对 GAD 特性的研究对掌握 GABA 的代谢具有重要意 义。本研究基于转录组数据库数据设计特异性引物，结合 RT-PCR 及 RACE 技术，扩增获 得柑橘全爪螨谷氨酸脱羧酶基因全长，命名为 PcGAD。PcGAD 基因全长为 1,991 bp，其开 放阅读框长度为 1,548 bp，能编码 515 个氨基酸。5’非编码区长度为 358 bp，3’非编码区 长度为 85 bp。预测理论等电点为 6.18，理论分子量为 59.3 kDa。序列分析发现，该基因具 有高度保守的中部区域，其中含有 GAD 酶的功能催化结构域，含有一个磷酸吡哆醛结合位 点 ser-x-x-lys。系统发育分析表明，PcGAD 与叶螨科的二斑叶螨谷氨酸脱羧酶亲缘关系最近。 此外，结合 RT-qPCR 分析不同发育阶段表达模式，旨在从分子水平初步明确 PcGAD 基因 的时空表达模式，为γ-氨基丁酸合成调控及柑橘全爪螨综合防治提供新思路。 关键词：柑橘全爪满；谷氨酸脱羧酶；cDNA 克隆；qPCR 中图分类号：请查阅《中国图书馆分类法》 20 Cloning and expression analysis of PcGAD of Panonychus citri XIA Wenkai, ZHONG Rui, DING Tianbo, LIAO Chongyu, DOU Wei, WANG Jinjun (Key Laboratory of Entomology and Pest Control Engineering, College of Plant Protection, 25 30 35 40 Southwest University, Chongqing 400716) Abstract: Glutamate decarboxylase (GAD) is an enzyme widely present in insects. In insects, GAD can catalyze decarboxylation of giutamate to formate of γ-aminobutyric acid. At the same time, GAD is the key enzyme in the biosynthesis of synthesis of GABA. Study on the characteristics of GAD has an important practical significance to exploringGABA metabolic. In this study, based on the transcriptome datasets, the full-length of PcGAD cDNA sequence was cloned by RT-PCR and RACE technology. The complete PcGAD cDNA consist 1,991 nucleotides with an ORF of 1,548 nucleotides which encodes 532 amino acid residues. The predicted molecular mass of the deduced PcGAD is about 59.3 kDa, while the isoeletric point is 6.18. PcGAD has a central conserved region, which contains a catalytic domain and a Pyridoxal phosphate binding site (ser-x-x-lys) of GAD. Phylogenetic analysis showed that PcGAD was most closely related to GAD from Tetranychus urticae. Moreover, RT-qPCR analysis of expression patterns during diff