意大利蜜蜂amLDH基因的克隆与表达分析

乳酸脱氢酶(LDH)是糖酵解和葡萄糖异生途径中一种发挥重要作用的蛋白酶。本研究以意大利蜜蜂为研究对象,RT-PCR和生物信息学方法克隆并分析了意大利蜜蜂乳酸脱氢酶基因及其表达产物的分子特征和结构功能,并利用qRT-PCR技术对amLDH基因在意大利蜜蜂不同级型、不同发育阶段的表达模式进行了分析。序列特征结果表明,意大利蜜蜂amLDH基因包含一个1 008 bp的开放阅读框,编码335个氨基酸,该氨基酸序列与中华蜜蜂高度相似(96.4%),并存在13个磷酸化位点、2个乳酸脱氢酶功能域,是一种稳定的疏水性蛋白。qRT-PCR结果表明,amLDH基因在不同级型、不同发育时期的表达差异显著(P<0.05)。工蜂卵期3日龄、雄蜂卵期3日龄、蜂王幼虫期4日龄表达最高,蜂王和雄蜂预蛹期至红眼蛹期表达均高于工蜂。本研究结果推测,amLDH基因在意大利蜜蜂的生长发育以及糖酵解中发挥重要作用,该研究结果能够为进一步探讨amLDH在意大利蜜蜂生殖发育中的功能提供理论依据。

Cloning and expression analysis of amLDH gene in Apis mellifera

Lactate dehydrogenase (LDH) plays an important role in glycolysis and gluconeogenesis of protease. To explore the molecular characteristics and expression patterns of amLDH gene, in this study, the amLDH genes was cloned by RT-PCR, and its expression patterns of various caste, as well as at different developmental stages of Apis mellifera were analyzed. Moreover, its sequence characteristics were determined by bioinformatics methods. Sequence analysis indicated that the amLDH gene of A. mellifera contained an opening reading frame of 1 008 bp, encoding 335 amino acids, the sequence of amLDH was highly homologous to that of Apis cerana(96.4%). The amLDH had 13 phosphorylation sites and 2 functional domains of lactate dehydrogenase, which was a stable hydrophobic protein. The results of qRT-PCR indicated that the expression level of amLDH gene was of significant difference in various castes and at different developmental stages of A. mellifera(P<0.05). Expression in workers was the highest at 3 days of age in the egg stage. The highest expression in drones and queen was in 3 day-old egg and 4 day-old larvae, respectively. Expression of queen bees and drones was higher than that of worker bees from prepupa to red-eye pupa stage. The results suggested that the amLDH gene played an important role in the growth and glycolysis. This study provided a theoretical basis for exploring the function of amLDH gene in reproductive development in A. mellifera.