棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析

doi:10.16380/j.kcxb.2019.07.003

摘要/Abstract

摘要：

【目的】棉铃虫Helicoverpa armigera的剂量补偿(dosage compensation, DC)分子机制尚不清楚。本研究旨在通过克隆棉铃虫雄性特异性致死(malespecific lethal, msl) 基因Hamsl1，利用RNA干扰技术明确其是否参与调控棉铃虫剂量补偿。【方法】利用RT-PCR同源克隆棉铃虫Hamsl1基因全长cDNA; 利用qPCR技术研究Hamsl1基因在棉铃虫不同发育时期的表达谱；通过显微注射Hamsl1 siRNA到棉铃虫3龄幼虫中对Hamsl1基因进行RNA干扰后，利用qPCR技术检测15个Z染色体基因的表达情况，分析Hamsl1是否调控Z染色体基因剂量。【结果】成功克隆了棉铃虫Hamsl1基因的cDNA序列，鉴定出Hamsl1基因mRNA存在2种剪接体，分别命名为Hamsl1a(GenBank登录号: MK564008)和Hamsl1b(GenBank登录号: MK564009)。功能域分析发现HaMSL1含有典型的PEHE和coiled-coil功能域，具有MSL1蛋白的特征。qPCR分析表明，Hamsl1基因位于棉铃虫Z染色体上；棉铃虫Hamsl1a与Hamsl1b基因表达均具有发育时期特异性，在成虫期表达量最高，且雌雄化蛹后基因表达量差异显著，具有性别特异性。通过同源比对和qPCR分析，在DNA水平鉴定了15个Z染色体候选基因。显微注射Hamsl1 siRNA于3龄幼虫体内72 h，干扰效率为36.01%~64.27%，并未发生雄性致死现象；与对照组相比，Hamsl1 RNAi处理组中棉铃虫15个Z染色体基因在雄性个体中整体呈现表达量上调趋势，而在雌性个体中平均表达水平差异不显著。【结论】本研究初步探明Hamsl1基因位于棉铃虫Z染色体上，且该基因可能通过抑制雄性棉铃虫Z染色体基因表达，调控棉铃虫Z染色体剂量补偿。本研究为深入研究棉铃虫剂量补偿分子机制和绿色防控棉铃虫提供了理论基础。

关键词: 棉铃虫, Hamsl1, 性染色体, 剂量补偿, RNAi

Abstract: 【Aim】 The dosage compensation mechanism of Helicoverpa armigera is still unclear. This study aims to clone male-specific lethal 1 gene Hamsl1 from H. armigera and to investigate whether it regulates dosage compensation of H. armigera by RNA interference technology. 【Methods】 The full-length cDNA of Hamsl1 sequence of H. armigera was cloned by RT-PCR based on homologous sequence alignment. The gene expression profiles of Hamsl1 in different developmental stages of H. armigera were detected by qPCR. After RNAi of Hamsl1 via microinjection of Hamsl1 siRNA into the 3rd instar larvae of H. armigera, the expression levels of 15 Z-linked genes were detected by qPCR to verify whether Hamsl1 regulates the dose of genes on Z chromosome. 【Results】 The full-length cDNA of Hamsl1 was successfully cloned from H. armigera, and its mRNA was identified to have two alternative splicing transcripts, which were designated as Hamsl1a (GenBank accession no.: MK564008) and Hamsl1b (GenBank accession no.: MK564009), respectively. Functional domain analysis showed that HaMSL1 contains a typical PEHE domain and a coiled-coil domain, which are the characteristics of MSL1 protein. The qPCR analysis demonstrated that Hamsl1 gene was located on the Z chromosome of H. armigera. The expression of both Hamsl1a and Hamsl1b showed the developmental stage specificity, peaking at the adult stage, and the sex specificity with significantly different expression level between female and male after pupation. Fifteen Z-linked genes were identified by homologous alignment and qPCR at the DNA level. Microinjecting Hamsl1 siRNA into the 3rd instar larvae of H. armigera for 72 h resulted in a RNA interference efficiency of 36.01%-64.27%, but no male specific lethal phenomenon was found in the tested insects. The 15 Z-linked genes in the Hamsl1 RNAi group showed significantly up-regulated expression in males as compared to those in the control group, but no significant expression change in females. 【Conclusion】 This study preliminarily proves that Hamsl1 gene is located on the Z chromosome of H. armigera and may regulate the dosage compensation by inhibiting the expression of Z-linked genes in male H. armigera. This study provides a theoretical foundation for further study of the molecular mechanism of dosage compensation and environmentally-friendly control of H. armigera.

Key words: Helicoverpa armigera, Hamsl1, sex chromosome, dosage compensation, RNAi

张亚坤, 邓中原, 谷少华, 李显春. 棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析[J]. 昆虫学报, 2019, 62(7): 799-813.

ZHANG Ya-Kun, DENG Zhong-Yuan, GU Shao-Hua, LI Xian-Chun. Identification and functional analysis of the dosage compensation related gene Hamsl1 in Helicoverpa armigera (Lepidoptera: Noctuidae)[J]. Acta Entomologica Sinica, 2019, 62(7): 799-813.

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棉铃虫剂量补偿相关基因Hamsl1的鉴定与功能分析

Identification and functional analysis of the dosage compensation related gene Hamsl1 in Helicoverpa armigera (Lepidoptera: Noctuidae)

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