›› 2018, Vol. 61 ›› Issue (10): 1237-1246.doi: 10.16380/j.kcxb.2018.10.013

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张贺贺, 张琪文, 林嘉, 季清娥, 陈家骅    

  1.   (福建农林大学益虫研究所, 联合国(中国)实蝇防控研究中心, 闽台作物有害生物生态防控国家重点实验室, 生物农药与化学教育部重点实验室, 福州 350002)
  • 出版日期:2018-10-20 发布日期:2018-10-20

Molecular cloning and expression profiling of bursicon genes in the fruit fly Zeugodacus tau (Diptera: Tephritidae)

ZHANG He-He, ZHANG Qi-Wen, LIN Jia, JI Qing-E, CHEN Jia-Hua*   

  1.  (UN (China) Center for Fruit Fly Prevention and Treatment, State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Key Laboratory of Biopesticide and Chemical Biology, Ministry of Education, Institute of Beneficial Insects, Fujian Agriculture and Forestry University, Fuzhou 350002, China)
  • Online:2018-10-20 Published:2018-10-20

摘要: 【目的】克隆南亚实蝇Zeugodacus tau鞣化激素基因,分析其分子特征及时空表达模式,为探索其生理功能奠定基础。【方法】利用同源克隆和RACE技术从南亚实蝇刚羽化成虫中克隆鞣化激素基因 bursicon-αbursicon-β的全长cDNA序列,并用邻接法(neighbor-joining method)与其他昆虫同源序列构建系统发育进化树。利用荧光定量PCR技术检测这两个基因在南亚实蝇不同发育阶段(卵、1-3龄幼虫、预蛹、蛹和刚羽化成虫)的表达特性。【结果】克隆获得南亚实蝇鞣化激素基因bursicon-α (GenBank登录号:MH421861)和bursicon-β (GenBank登录号:MH421862)。bursicon-α基因开放阅读框为 551 bp,编码183个氨基酸;bursicon-β基因开放阅读框为467 bp,编码156个氨基酸。基于两个鞣化激素基因的氨基酸序列的系统发育树分析显示,南亚实蝇Bursicon-α与Bursicon-β均与瓜实蝇 Zeugodacus cucurbitae的同源蛋白亲缘关系最近,且与其他双翅目昆虫的同源蛋白聚为一类,形成独立的分支。荧光定量PCR结果表明,两个基因在南亚实蝇各龄期均有表达,均在第5天蛹和刚羽化成虫翅伸展时期表达量最高。【结论】bursicon-αbursicon-β基因在南亚实蝇不同发育阶段表达量不同,推测其在南亚实蝇成虫表皮鞣化和翅的形成中发挥着重要作用。本研究为进一步探索鞣化激素在南亚实蝇生长过程中表皮的骨化、翅的重建等方面的功能机制奠定了基础。

关键词: 南亚实蝇, 鞣化激素, 表皮骨化, 基因表达, 翅伸展, 系统发育

Abstract: 【Aim】 To clone bursicon genes from the fruit fly Zeugodacus tau and to analyze their molecular characteristics and spatiotemporal expression patterns, so as to develop a basis for further study of their physiological functions. 【Methods】 The complete cDNAs of bursicon-α and bursicon-β genes were cloned from the newly emerged adults of Z. tau by using homology-based cloning and RACE technology, and the phylogenetic trees were constructed with the homologous sequences of other insect species by using neighbor-joining method. The expression patterns of the two genes in different developmental stages (egg, 1st-3rd instar larva, prepupa, pupa and newly emerged adult) of Z. tau were detected by qPCR. 【Results】 The full-length cDNAs of two bursicon genes bursicon-α (GenBank accession no.: MH421861) and bursicon-β (GenBank accession no.: MH421862) were cloned from Z. tau. bursicon-α contains a 551 bp open reading frame (ORF) encoding 183 amino acid residues, and bursicon-β has a 467 bp ORF encoding 156 amino acid residues. Phylogenetic tree analysis based on the amino acid sequences of the two bursicon genes showed that Bursicon-α and Bursicon-β of Z. tau had a closest genetic relationship with the homologous proteins of Zeugodacus cucurbitae, and clustered into one independent group with the homologous proteins of other dipterous insects. The qPCR results indicated that both bursicon-α and bursicon-β were expressed in all developmental stages of Z. tau, and highly expressed in 5 d-old pupae and in the wing expansion stage of the newly emerged adults. 【Conclusion】 The expression levels of bursicon-α and bursicon -β are different in different developmental stages of Z. tau, suggesting a crucial role of bursicon genes in cuticular tanning and wing formation. This study provides a foundation for further exploring the functional mechanisms of bursicon in cuticle sclerotization and wing reconstruction of Z. tau.

Key words: Zeugodacus tau, bursicon, cuticle sclerotization, gene expression, wing expansion, phylogeny