意大利蜜蜂amLDH基因的克隆与表达分析

doi:10.3969/j.issn.1004-1524.2021.11.07

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (11): 2051-2058.DOI: 10.3969/j.issn.1004-1524.2021.11.07

意大利蜜蜂amLDH基因的克隆与表达分析

欧阳霞辉(), 郑天宇, 徐文凯, 郑相相

西北民族大学生命科学与工程学院,甘肃兰州 730030

收稿日期:2020-08-16 出版日期:2021-11-25 发布日期:2021-11-26
作者简介:欧阳霞辉 (1978—),女,甘肃兰州人,博士,副教授,主要从事昆虫生殖与发育方面的研究。E-mail: oyxhui316@126.com
基金资助:
国家自然科学基金(31660628);甘肃省自然科学基金(1606RJZA058);中央高校基本科研业务费专项资助项目(31920210128)

Cloning and expression analysis of amLDH gene in Apis mellifera

OUYANG Xiahui(), ZHENG Tianyu, XU Wenkai, ZHENG Xiangxiang

College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China

Received:2020-08-16 Online:2021-11-25 Published:2021-11-26

摘要/Abstract

摘要：

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。

关键词: 乳酸脱氢酶, 生物信息学分析, 意大利蜜蜂, 荧光定量

Abstract:

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.

Key words: lactate dehydrogenase, bioinformatics method, Apis mellifera, fluorescence quantification

中图分类号:

S891

欧阳霞辉, 郑天宇, 徐文凯, 郑相相. 意大利蜜蜂amLDH基因的克隆与表达分析[J]. 浙江农业学报, 2021, 33(11): 2051-2058.

OUYANG Xiahui, ZHENG Tianyu, XU Wenkai, ZHENG Xiangxiang. Cloning and expression analysis of amLDH gene in Apis mellifera[J]. Acta Agriculturae Zhejiangensis, 2021, 33(11): 2051-2058.

图/表 7

表1 本研究所用引物信息

Table 1 Primers of this study

引物名称 Primer name	引物序列 Primer sequence (5'-3')
LDH-F	AGAACGGCTGGTAAACCGTC
LDH-R	CGAACGATGAACGGATTCGG
q-LDH(s)	ATTTGGTGTTTGCTTCATTCTCTT
q-LDH(a)	CTTCTCAACGATGCCATTATTCT
qβ-actin-2F	TGCCAACACTGTCCTTTCTG
qβ-actin-2R	AGAATTGACCCACCAATCCA

图1 意大利蜜蜂amLDH基因PCR扩增结果 M,DL2000 DNA marker;1,以水为模板的阴性对照;2,amLDH基因PCR产物。

Fig.1 PCR amplification of amLDH gene in Apis mellifera M, DL2000 DNA marker; 1, A negative control with water as the template; 2, PCR products of amLDH gene.

图2 采用NJ法构建的amLDH基因系统发育树

Fig.2 Phylogenetic tree of amLDH gene based on NJ method

表2 意大利蜜蜂amLDH的理化性质

Table 2 Physicochemical properties of amLDH in Apis mellifera

蛋白质理化特性 Physicochemical properties of proteins	预测结果 Prediction results
分子式Molecular formula	C₁₆₁₈H₂₆₄₁N₄₃₉O₄₉₆S₁₃
等电点Isoelectric point	6.66
负电荷氨基酸残疾总数(Asp + Glu)Total disability of negatively charged amino acids(Asp + Glu)	35
正电荷氨基酸残疾总数(Arg + Lys)Total disability of positive charge amino acids(Arg + Lys)	34
不稳定指数Instability index	29.67
总平均亲水性系数 Total average hydrophilicity coefficient	0.005
半衰期Half life/h	30

图3 预测的amLDH蛋白三级结构

Fig.3 Predicted tertiary structure of amLDH protein

图4 amLDH基因在意大利蜜蜂工蜂、雄蜂、蜂王中的表达柱状图上同个系列无相同小写字母表示经单因素方差分析各个时期组间差异显著(P<0.05,Duncan氏多重比较法)。

Fig.4 The expression of amLDH gene in Apis mellifera worker bees, drones and queen bees The bars without the same lower case letters in the same series showed significant differences among groups in different periods by one-way ANOVA (P<0.05, Duncan’s Multiple Comparison).

图5 amLDH基因在不同级型、不同发育阶段的意大利蜜蜂中的表达情况 L,卵期;F,孵化期;YD,幼虫第一日;YZ,幼虫最后一日;Y,预蛹;B,白眼蛹;H,红眼蛹;C,成虫。

Fig.5 The expression of amLDH gene in different castes and different development stages of Apis mellifera L, Egg stage; F, Hatching period; YD, First day of larval; YZ, Last day of larva; Y, Prepupa; B, White-eyed pupa; H, Red-eyed pupa; C, Adult.

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意大利蜜蜂amLDH基因的克隆与表达分析

Cloning and expression analysis of amLDH gene in Apis mellifera

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