Cloning and expression analysis of amLDH gene in Apis mellifera

doi:10.3969/j.issn.1004-1524.2021.11.07

Acta Agriculturae Zhejiangensis ›› 2021, Vol. 33 ›› Issue (11): 2051-2058.DOI: 10.3969/j.issn.1004-1524.2021.11.07

• Animal Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S891

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.

Figures/Tables 7

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

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.

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

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

Fig.3 Predicted tertiary structure of amLDH protein

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).

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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Cloning and expression analysis of amLDH gene in Apis mellifera

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