中华蜜蜂foraging基因Acfor的 克隆与发育差异表达
Cloning and expression analysis of the foraging gene in the Chinese honeybee (Apis cerana cerana Fabricius)
马卫华1, 2** 邵有全1 赵慧婷3 孟 娇2 田嵩浩4 杜亚丽2 姜玉锁2***
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DOI：10.7679/j.issn.2095-1353.2017.007
作者单位：1. 山西农业科学院园艺研究所，太原 030031；2. 山西农业大学动物科技学院，太谷 030801； 3. 山西农业大学生命科学学院，太谷 030801；4. 山西医科大学汾阳医学院，汾阳 032200
中文关键词：中华蜜蜂，foraging，基因克隆，发育表达模式，PKG活性
英文关键词：Chinese honeybee, foraging gene, cloning, development expression pattern, PKG activity
中文摘要：
【目的】 本研究克隆了中华蜜蜂Apis cerana cerana Fabricius foraging（Acfor）基因，并对其进行了序列和基因表达分析，为研究该基因参与蜜蜂劳动分工的分子机理及蜜蜂采集行为调控奠定基础。 【方法】 利用RT-PCR技术扩增和克隆获得Acfor的全长序列，采用生物信息学软件对其蛋白进行结构特点分析；采用qRT-PCR对Acfor基因的表达特性进行分析；环鸟苷酸依赖的蛋白激酶（PKG）活性采用比色法检测。【结果】 Acfor cDNA 全长为3 029 bp（GenBank登录号为，KP662686.1），ORF序列长度为2 169 bp，编码722个氨基酸。预测该蛋白分子量为81.80 ku，理论等电点（PI）5.50，为酸性、稳定的、亲水性蛋白，无信号肽和跨膜结构，存在2个糖基化位点和38个潜在磷酸化位点，主要分布在细胞质中；二级结构中，有卷曲环、α-螺旋和伸展链3种结构，三者比例分别为57.06%、28.12%和14.82%；系统发育树结果显示，中华蜜蜂Acfor和其它膜翅目for组成了一个大的分支，分支由7个亚支构成，Acfor与Amfor分子距离最近。不同日龄工蜂Acfor mRNA的表达和PKG活性的趋势一致，1~30日龄均有表达和PKG活性，1~10日龄表达量和活性下降，10日龄后开始上升，25日龄表达量和活性最高，然后随着日龄的增加而下降。【结论】 可见，Acfor基因表达和PKG活性水平影响中华蜜蜂与日龄有关的采集行为。
英文摘要：
[Objectives] To lay a foundation for studying the molecular mechanisms by which bee genes regulate the division of labor and foraging behavior. [Methods] The cDNA sequence of Acfor from the brain of Apis cerana cerana Fabricius was cloned using the Reverse transcription-polymerase chain reaction (RT-PCR) and its protein structure analyzed using bioinformatics software. A quantitative analysis of its expression in worker bees on successive days was conducted by the quantitative real-time PCR (qRT–PCR). The activity of cGMP-dependent protein kinase (PKG) was quantified with the colorimetric method. [Results] The results show that the full-length cDNA sequence of Acfor is 3 029 bp (GeneBank accession no.KP662686.1), including an open reading frame (ORF) of 2 169 bp that encodes 722 amino acids. The molecular weight and predicted isoelectric point of this protein are 81.80 ku and 5.50, respectively. The protein is water-soluble, acidic and stable, has no signal peptide or transmembrane domain, and has 2 glycosylation sites and 39 phosphorylation sites that may be located in the cytoplasm. The predicted secondary structure shows that Acfor protein is comprised of 57.06% random coil, 28.12% alpha helix and 14.82% extended strand. Phylogenetic analysis indicates that Acfor of A.c.cerana is in the same clade as similar proteins of other Hymenopteran insects. This clade has seven sub-branches, and the protein with the closest molecular distance to Acfor is Amfor. Acfor transcript expression and PKG activity were detected in the heads of workers of different ages. Temporal trends of Acfor mRNA expression and PKG activity were similar. Gene expression and PKG activity decreased from day 1 to 10, and begin to rise after day 10, remaining at peak levels until day 25 after which they declined with increasing age. [Conclusion] Acfor expression and PKG activity levels affect A. c. cerana age-related foraging behavior.