棉铃虫鞣化激素基因克隆及分子特性分析

【目的】获得棉铃虫鞣化激素2个亚基(α亚基和β亚基)基因序列,分析其分子特性和表达模式,研究棉铃虫鞣化激素的作用机制奠定基础。【方法】通过生物信息学和分子生物学技术分别得到棉铃虫鞣化激素α和β亚基cDNA序列,将棉铃虫及NCBI中公布的已知其它昆虫鞣化激素α和β亚基氨基酸序列分别整理分析,利用MEGA7(7.0.14)软件的Jones-Taylor-Thornton(JTT)模型构建系统进化树并进行聚类分析,qRT-PCR分别检测两亚基在棉铃虫不同生长发育阶段的表达模式。【结果】分别获得了棉铃虫鞣化激素 α亚基与β亚基核苷酸序列。其中α亚基(GenBank登录号:AHM0247472.1)cDNA片段长694 bp,开放阅读框480 bp,编码159个氨基酸残基,预测该蛋白质分子量为17.7 kDa。该基因在卵期第3 d、1龄幼虫第1 d、3~5龄幼虫第1 d、2和3龄末蜕皮期(3M和4M)、蛹期第0 d、第3 d和第7 d表达量相对较高。β亚基(GenBank登录号:AHM0247473.1)cDNA片段长779 bp,开放阅读框420 bp,编码139个氨基酸残基,预测该蛋白质分子量为15.9 kDa。该基因在卵期至2龄末蜕皮期(3M)、3龄幼虫第2 d、4~5龄幼虫第1 d、蛹期第1 d至羽化前表达量相对较高。鞣化激素α亚基和β亚基基因均在棉铃虫胸神经节中相对表达量最高,咽下神经节次之,脑部和腹部相对表达量较弱。【结论】鞣化激素在鳞翅目昆虫中具有较高的保守性;α亚基和β亚基基因主要在棉铃虫卵期、初孵幼虫期、蛹期和新羽化成虫期发挥作用;鞣化激素在棉铃虫幼虫体内主要由胸部神经节转录合成。

Gene Cloning and Molecular Characterization of Bursicon Gene in Helicoverpa armigera(Lepidoptera: Noctuidae)

【Objective】 Bursicon, a kind of neuropeptitde hormone, consists of heterogenous proteins, and plays an important role in the cuticle development and sclerotization of inscets. This study aims to obtain the cDNA sequence of two subunits of bursicon(α subunit as burs and β subunit as pburs). And we analyzed its molecular characteristics and expression pattern of bursicon of Helicoverpa armigera, in order to provide a foundation for further functional mechanism.【Methods】 The full-length cDNA sequence of bursicon gene was cloned from H. armigera by using bioinformatics and molecular cloning technique. The phylogenetic tree was constructed based on the amino sequences of burs and pburs of H. armigera respectively compared with other published and known insects of burs and pburs. And cluster analysis was operated with Jones Taylor Thornton (JTT) model of mega7 (7.0.14) software. The developmental expression patterns of two genes were respectively assayed with qRT-PCR.【Results】 We cloned the full-length cDNA sequence of burs (GenBank accession no: AHM0247472.1) and pburs(β subunit) (GenBank accession no: AHM0247473.1) from H. armigera, the α subunit(burs) is 694 bp in length with a 480 bp open reading frame encoding 561 amino acids. Burs relatively highly expressed in day-3 of egg stage, day-1 of the 1st instar larval stage, day-1 of the 3rd-5th instar larval stages, 3rd-4th instar molting stages, day-0, day-3, day-7 of prepupal stages. The pburs is 779 bp in length with a 420 bp open reading frame encoding 139 amino acids. The β subunit (pburs) relatively highly expressed from egg stages to 3rd instar molting stages, day-2 of the 3rd instar larval stage, day-1 of the 4th-5th instar larval stages, day-1 of prepupal stage before adult. In addition, burs and pburs have a higher expression level in the thoracic ganalia of cotton bollworms, the second in the hypopharyngeal ganglia, and the lower in the brain and abdominal ganalia.【Conclusion】 Bursicon has a higher conservation in Lepidoptera, burs and pburs mainly act on the egg stage, instar larva stage, pupa stage and emerging adult stage. Bursicon is mainly synthesized by thoracic ganglia in the larva of cotton bollworm.