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