›› 2018, Vol. 61 ›› Issue (2): 178-187.doi: 10.16380/j.kcxb.2018.02.004

• 研究论文 • 上一篇    下一篇


李鸿波, 戴长庚, 张昌容, 何永福*, 胡阳*   

  1. (贵州省农业科学院植物保护研究所, 贵阳 550006)
  • 出版日期:2018-02-20 发布日期:2018-02-20

Cloning and expression profiling of catalase gene in the oriental armyworm, Mythimna separata (Lepidoptera: Noctuidae)

LI Hong-Bo, DAI Chang-Geng, ZHANG Chang-Rong, HE Yong-Fu*, HU Yang*    

  1.  (Institute of Plant Protection, Guizhou Academy of Agricultural Sciences, Guiyang 550006, China)
  • Online:2018-02-20 Published:2018-02-20

摘要:  【目的】过氧化氢酶(CAT)的主要功能是将过氧化氢分解成水和氧气,从而使生物免受氧化损伤的伤害。本研究旨在探索CAT在粘虫Mythimna separata抗氧化胁迫中的作用。【方法】采用RT-PCR和RACE技术克隆粘虫CAT基因的cDNA全长序列和基因组序列,利用生物信息学软件分析该基因及其编码蛋白质的序列特性;运用实时荧光定量PCR(qPCR)技术分析该基因在不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(头、表皮、前肠、中肠、后肠和马氏管)以及毒死蜱、高温和密度胁迫下幼虫中的表达谱。【结果】克隆得到的粘虫CAT基因命名为MsCAT(GenBank登录号: MF737386),其全长cDNA长1 846 bp,开放阅读框为1 602 bp,编码533个氨基酸。序列分析发现MsCAT具有CAT家族典型的结构域,包括一个基部活化位点标签(88FDRERIPERVVHAKGAGA105),一个NADPH结合位点(216VTHQVLYLFGD226)和一个亚铁血红蛋白结合位点(379RLFSYSDTH386)。DNA序列分析表明,在MsCAT编码区99 bp处插入了一个612 bp的内含子。系统发育分析显示,MsCAT与夜蛾科(Noctuidae)昆虫的亲缘关系最近。MsCAT在粘虫的各个发育阶段和5龄幼虫各组织中均表达,分别在6龄幼虫和幼虫中肠中的表达量最高。取食经1 μg/mL毒死蜱处理的玉米叶片24 h后,幼虫体内MsCAT表达量显著上调,但随浓度提高,表达量反而下降;在33~37℃温度处理后,幼虫MsCAT的表达量显著高于对照(24℃),而39℃处理幼虫MsCAT的表达量下调;此外,随饲养密度的增加幼虫体内MsCAT的表达显著下调。【结论】本研究克隆得到的粘虫CAT基因的cDNA全长序列(MsCAT)是可靠的,并且MsCAT在粘虫发育及抗氧化胁迫中具有重要的作用。

关键词: 粘虫, 过氧化氢酶, 基因克隆, 表达谱, 氧化胁迫, 实时荧光定量PCR

Abstract: 【Aim】 Catalase (CAT) plays an important role in decomposing hydrogen peroxide into water and oxygen which makes an organism avoiding the damage of oxidative stress. This study aims to explore the roles of CAT in anti-oxidative stress in the oriental armyworm, Mythimna separata. 【Methods】 The complete cDNA and genomic sequence of CAT in M.separata were cloned by RT-PCR and RACE. Bioinformatics programs were used to analyze the sequence characteristics of the gene and encoded protein. The expression levels of CAT in M. separata in different developmental stages (egg, 1st-6th instar larva, pupa and adult), tissues of the 5th instar larva (head, cuticle, foregut, midgut, hindgut and Malpighian tubules), and larvae under chlorpyrifos exposure, high temperature stress and larval crowding conditions were detected by quantitative real-time PCR (qPCR). 【Results】 The complete cDNA of CAT obtained from M. separata was named as MsCAT (GenBank accession no.: MF737386), which is 1 846 bp in length, with a 1 602 bp opening reading frame (ORF) encoding 533 amino acids. Sequence analysis indicated that MsCAT has three typical motifs of CAT family, including one proximal active site (88FDRERIPERVVHAKGAGA105), one NADPH binding site (216VTHQVLYLFGD226)and one proximal heme-ligand signature sequence (379RLFSYSDTH386). The DNA sequence of MsCAT contains a 612 bp intron that inserts into the location behind the 99th bp of the encoding region. Phylogenetic analysis indicated that CATs from Noctuidae moths could be assigned to one well-supported cluster. MsCAT was expressed in various developmental stages and tissues of the 5th instar larva of M. separata, and exhibited the highest expression level in the 6th instar larval stage and larval midgut, respectively. MsCAT was significantly up-regulated in larvae fed on corn leaves treated by low concentration (1 μg/mL) of chlorpyrifos for 24 h, but down-regulated in larvae fed on high concentrations of chlorpyrifos. The expression level of MsCAT was significantly up-regulated in larvae exposed to temperatures from 33 to 37℃, but declined in larvae exposed to 39℃ as compared with that of the control (24℃). The expression level of MsCAT in M. separata larvae was negatively related to their crowding degree. 【Conclusion】 The obtained full-length cDNA of MsCAT encoding CAT in M. separata is reliable, and MsCAT may play an important role in development and oxidative stress tolerance of M. separata.

Key words: Mythimna separata, catalase, gene cloning, expression profiles, oxidative stress, quantitative real-time PCR