中国农业科学 ›› 2013, Vol. 46 ›› Issue (8): 1706-1715.doi: 10.3864/j.issn.0578-1752.2013.08.021

• 畜牧·资源昆虫 • 上一篇    下一篇

中华蜜蜂化学感受蛋白基因家族克隆及表达特征分析

 倪翠侠, 张林雅, 李红亮, 商晗武   

  1. 中国计量学院生命科学学院/浙江省生物计量及检验检疫技术重点实验室,杭州 310018
  • 收稿日期:2012-10-19 出版日期:2013-04-15 发布日期:2012-11-19
  • 通讯作者: 通信作者李红亮,Tel:0571-86835774;E-mail:hlli@cjlu.edu.cn
  • 作者简介:倪翠侠,Tel:13588134372;E-mail:ncx1208@163.com
  • 基金资助:

    国家自然科学基金(30900163)

Molecular Cloning and Expression Profiles Analysis of Chemosensory Protein Genes Family in the Chinese Honeybee (Apis cerana cerana)

 NI  Cui-Xia, ZHANG  Lin-Ya, LI  Hong-Liang, SHANG  Han-Wu   

  1. College of Life Sciences, China Jiliang University/Key Laboratory of Biometrology and Inspection& Quarantine of Zhejiang Province, Hangzhou 310018
  • Received:2012-10-19 Online:2013-04-15 Published:2012-11-19

摘要: 【目的】完成中华蜜蜂(Apis cerana cerana)化学感受蛋白(chemosensory proteins,CSPs)基因家族的序列及表达谱鉴定,解析中蜂整个CSPs家族的表达特征与分布规律,为中蜂CSPs的生物学功能研究提供参考。【方法】利用RT-PCR技术扩增,克隆获得中蜂CSPs基因家族全部6条基因序列的开放阅读框(ORF)全长,并利用real-time PCR技术对中蜂所有的CSPs序列在中蜂工蜂不同发育历期及不同器官表达特征进行研究。【结果】新克隆4条基因(Ac-CSP2、Ac-CSP4、Ac-CSP5和Ac-CSP6)的开放阅读框分别为354、387、315和378 bp,分别编码117、128、104和125个氨基酸残基,预测蛋白质分子量分别为13.01、14.61、12.46和14.63 kD,等电点分别为8.86、4.53、9.60和8.71,且均具有4个保守的半胱氨酸残基,均符合昆虫CSPs的一般生化特征。中蜂与意蜂CSPs同源基因家族间具有高达95%—99%的相似性。CSPs在中蜂不同发育时期及器官的表达特征结果显示,Ac-CSP1、2、3、4在卵、幼虫和蛹期几乎不表达,其中Ac-CSP1、2、4在触角中表达量最高,而Ac-CSP3在翅中表达量最高;Ac-CSP5在中蜂各个发育历期的表达量几乎相同;Ac-CSP6蛹期有较高表达,其余时期不表达。【结论】中蜂CSPs基因家族所有6个基因的氨基酸序列与意大利蜜蜂有较高的相似性,但二者各基因的表达谱在整体相似的情况下局部又存在着某些差异,为进一步研究该家族蛋白的生理功能机制奠定了良好基础。

关键词: 中华蜜蜂 , 化学感受蛋白 , 克隆 , DNA序列分析 , 表达谱

Abstract: 【Objective】 The objective of this study is to clone the whole genes family of chemosensory protein in the Chinese honeybee (Apis cerana cerana) (Ac-CSPs) and analyze their expression of mRNA in different tissues and different development time-points. This may provide a fundamental evidence for the future study of the physiological function of Ac-CSPs. 【Method】 Six full-length of CSP genes of A. cerana cerana was cloned by using reverse transcription-polymerase chain reaction (RT-PCR). The expression patterns at development phases and in different sensory organs of the six Ac-CSPs were analyzed by real-time PCR. 【Result】 Four full-length of Ac-CSP genes (Ac-CSP2, Ac-CSP4, Ac-CSP5 and Ac-CSP6) were cloned in which Ac-CSP2 was 354 bp in full length that encoded a polypeptide containing 117 amino acids with prediction of MW (molecular weight) of 13.01 kD and pI of 8.86, respectively. Ac-CSP4 was 387 bp in full length that encoded a polypeptide containing 128 amino acids with prediction of MW of 14.61 kD and pI of 4.53, respectively. Ac-CSP5 was 315 bp in full length that encoded a polypeptide containing 104 amino acids with prediction of MW of 12.46 kD and pI of 9.60, respectively. Ac-CSP6 was 378 bp in full length that encoded a polypeptide containing 125 amino acids with prediction of MW of 14.63 kD and pI of 8.71, respectively, and all of the genes with four conserved cysteine residues. The six deduced Ac-CSPs sequences showed high sequence similarity (95%-99%) as compared with CSPs (Am-CSPs) sequences of A. mellifera. The genes of Ac-CSP1, 2, 3, 4 were not expressed in eggs, larvae and pupae, with the Ac-CSP1, 2, 4 expressed at highest level in antennae and Ac-CSP3 was strongly expressed in wings. The expression of Ac-CSP5 was not significantly different at all developmental stages, while Ac-CSP6 only highly expressed in the pupae. 【Conclusion】 The alignment is very similar of Ac-CSPs and Am-CSPs and the expression patterns are similar but there is a part of difference in this research, which provides useful information for the study of function of CSPs in the future.

Key words: Apis cerana cerana , chemosensory proteins , molecular cloning , DNA sequence analysis , global expression