意大利蜜蜂amPFK基因的克隆与表达分析
Cloning and expression of the amPFK gene in Apis mellifera
郑相相；徐文凯；刘丽霞；欧阳霞辉
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DOI：10.7679/j.issn.2095-1353.2021.012
作者单位：西北民族大学，生命科学与工程学院，兰州 730030；西北民族大学，生命科学与工程学院，兰州 730030；西北民族大学，生命科学与工程学院，兰州 730030；西北民族大学，生命科学与工程学院，兰州 730030
中文关键词：意大利蜜蜂；磷酸果糖激酶；分子特征；表达模式；生殖调控
英文关键词： Apis mellifera; phosphofructokinase; molecular characteristics; expression pattern; reproductive regulation
中文摘要：
【目的】 本研究旨在明确意大利蜜蜂磷酸果糖激酶（amPFK）基因的序列特征及表达模式，为进一步研究amPFK在生殖和发育中的功能奠定理论基础。【方法】 采用RT-PCR技术克隆了amPFK基因，并对其氨基酸序列和蛋白结构进行了分析；通过 RT-qPCR 检测了amPFK基因在意大利蜜蜂不同品级不同发育时期的表达模式。【结果】 克隆获得了意大利蜜蜂amPFK的全长cDNA序列，其中开放阅读框（ORF）2 486 bp，编码757个氨基酸。序列分析表明，amPFK在进化过程中具有较高的保守性，可能存在两个磷酸果糖激酶结构域；amPFK蛋白主要由α-螺旋构成，是一个稳定的亲水性蛋白，具有1个可能的互作蛋白Vha44。表达模式显示，amPFK基因在意大利蜜蜂不同品级、不同发育时期的表达量差异显著(P˂0.05)；雄蜂、蜂王不同时期amPFK的表达量均呈现出先升高后降低再升高的趋势，羽化成虫后表达量最高；不同品级的幼虫第一日龄amPFK的表达量明显较高，而且羽化成虫后蜂王和雄蜂的表达量明显高于工蜂。【结论】 意大利蜜蜂amPFK在蜂王、雄蜂的幼虫初期和成虫期的高表达量可能与其胚后发育初期和生殖发育中细胞增殖的能量代谢有关。
英文摘要：
[Objectives] To provide a theoretical basis for research on the function of the amPFK gene in Apis mellifera. [Methods] The cDNA sequence of the amPFK gene was cloned by RT-PCR, and its amino acid sequence and protein structure analyzed using bioinformatic methods. RT-qPCR was used to detect the expression of the amPFK gene in different castes and developmental stages of A. mellifera. [Results] The open reading frame (ORF) of the amPFK gene is 2 486 bp, encoding 757 amino acids. Sequence analysis indicates that amPFK has been highly conserved, and may have two phosphate fructose kinase domains. The amPFK protein is mainly composed of an α-helix, which is a stable hydrophilic protein that may interact with Vha44. RT-qPCR indicated that expression of the amPFK gene is significantly different in different castes and developmental stages. The expression of ampfk in different drone and queen age classes tended to first increase, decrease, then increase. Expression was highest in adults. Among larvae of different ages expression of ampfk was significantly higher in one day-old larvae, and expression in queens and drones was significantly higher than that in workers. [Conclusion] The relatively high expression of amPFK in the early larval and adult stages of queens and drones may be related to the energy metabolism associated with cell proliferation in the early stages of postembryonic and reproductive development.