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棉铃虫BR-C Z4基因的克隆及表达分析

引用本文：刘静,张连军,胡得琴,张亚林,刘小宁.棉铃虫BR-C Z4基因的克隆及表达分析.植物保护学报,2023,50(4):962-971

DOI：10.13802/j.cnki.zwbhxb.2023.2022043

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作者	单位	E-mail
刘静	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830017
张连军	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830017
胡得琴	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830017
张亚林	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830017
刘小宁	新疆大学生命科学与技术学院, 新疆生物资源基因工程重点实验室, 乌鲁木齐 830017	liuxn0103@xju.edu.cn

中文摘要:为明确转录因子广泛锌指复合物（broad-complex，BR-C）Z4亚型在棉铃虫Helicoverpa armigera生长发育过程中的作用，基于2-十三烷酮（2-tridecanone，2-TD）处理的棉铃虫幼虫中肠转录组数据克隆获得BR-C Z4的开放阅读框（open reading frame，ORF）序列，对其氨基酸序列和编码蛋白质进行生物信息学分析，并利用实时荧光定量PCR（real-time fluorescence quantitative PCR，qPCR）技术分析BR-C Z4基因在棉铃虫不同龄期、不同组织和2-TD处理6龄幼虫中的表达规律以及其和Ⅳ型几丁质酶（chitinase IV，CHT4）编码基因在4~6龄初、末期幼虫中的表达规律。结果表明，克隆获得的棉铃虫BR-C Z4基因的ORF为1 347 bp，编码448个氨基酸，预测编码蛋白质的分子量和理论等电点分别为49.8 kD和7.75，且主要定位于细胞核中。系统进化分析结果显示，棉铃虫BR-C Z4与家蚕Bombyx mori和烟草天蛾Manduca sexta的BR-C Z4亲缘关系最近。BR-C Z4基因在棉铃虫整个生长期均有表达，在3龄期的相对表达量最低而在预蛹期的相对表达量最高；该基因在棉铃虫6龄幼虫的脂肪体、中肠、体壁和头部均有表达，在头部的相对表达量最低，在中肠中的相对表达量最高； BR-C Z4和CHT4基因在棉铃虫4~6龄末期幼虫中的相对表达量均显著高于4~6龄初期幼虫；相关性分析结果显示BR-C Z4和CHT4的相对表达量呈极显著正相关；随着2-TD处理浓度的增加，棉铃虫BR-C Z4基因的相对表达量总体呈现先上升后下降的趋势，其中15 mg/g浓度处理20 h时BR-C Z4基因相对表达量达到峰值，而20 mg/g浓度处理6 h时BR-C Z4基因相对表达量降到最低，为对照的22.73%。推测BR-C Z4基因在棉铃虫变态发育和应对2-TD胁迫过程中发挥着重要作用。

中文关键词:棉铃虫广泛锌指复合物克隆时空表达 2-十三烷酮

Cloning and expression analysis of BR-C Z4 gene in cotton bollworm Helicoverpa armigera

Author Name	Affiliation	E-mail
Liu Jing	Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, Xinjiang UygurAutonomous Region, China
Zhang Lianjun	Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, Xinjiang UygurAutonomous Region, China
Hu Deqin	Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, Xinjiang UygurAutonomous Region, China
Zhang Yalin	Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, Xinjiang UygurAutonomous Region, China
Liu Xiaoning	Xinjiang Key Laboratory of Biological Resources and Genetic Engineering, College of Life Science and Technology, Xinjiang University, Urumqi 830017, Xinjiang UygurAutonomous Region, China	liuxn0103@xju.edu.cn

Abstract:To investigate the role of transcription factor broad-complex (BR-C) subtype Z4 in the growth and development of cotton bollworm Helicoverpa armigera, the open reading frame (ORF) sequence of BR-C Z4 was cloned from larval midgut based on the transcriptomic data, which was obtained from midgut of H. armigera treated with 2-tridecanone (2-TD), and its amino acid sequence and protein structure were analyzed with bioinformatics. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression profiles of BR-C Z4 gene in different developmental stages, different tissues and its expression level in the 6th instar larvae treated with 2-TD, and the expression profiles of BR-C Z4 and chitinase IV (CHT4) genes in early stages and late stages of 4th-6th instar larvae. The results showed that the ORF of BR-C Z4 gene in H. armigera was 1 347 bp in length, encoding 448 amino acids. The predicted molecular weight and the isoelectric point of the BR-C Z4 protein were 49.8 kD and 7.75, and it was mainly located in the nucleus of H. armigera. Phylogenetic analysis showed that BR-C Z4 of H. armigera was closely related to BR-C Z4 of Bombyx mori and Manduca sexta. BR-C Z4 gene was expressed in all growth periods of H. armigera, and the expression level was lowest in the 3rd instar larvae and highest in the pre-pupa stage. BR-C Z4 gene was expressed in fat body, midgut, integument and head of 6th instar larvae of H. armigera, with the lowest expression level in head and the highest in midgut. The expression levels of BR-C Z4 gene and CHT4 gene from the late stage from 4th to 6th instar larvae were correspondingly higher than those from the early stage. There was a significant positive correlation between the expressions of BR-C Z4 and CHT4. The relative expression of BR-C Z4 gene of H. armigera treated with 2-TD increased firstly and then decreased, and the relative expression level of BR-C Z4 gene of H. armigera treated with 15 mg/g 2-TD for 20 h reached the peak. The relative expression level of BR-C Z4 gene decreased to 22.73% of the control group at 6 h after treatment with 20 mg/g 2-TD. It indicated that BR-C Z4 gene might play an important role in the metamorphosis development and response to 2-TD in H. armigera.

keywords:Helicoverpa armigera broad-complex (BR-C) cloning temporal and spatial expression 2-tridecanone (2-TD)

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