意大利蜜蜂amPGAM2基因的克隆、序列特征及表达分析 *

doi:10.7679/j.issn.2095-1353.2020.091

摘要/Abstract

摘要：

【目的】 磷酸甘油酸变位酶（PGAM）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,本研究拟明确amPGAM2基因的序列特征及表达模式。【方法】 以意大利蜜蜂Apis mellifera为研究对象,克隆了amPGAM2基因的cDNA序列,分析了其序列特征及其在工蜂、雄蜂、蜂王的不同发育时期的表达模式。【结果】 序列特征分析表明,克隆所得序列全长976 bp,包含1个完整的开放阅读框,共编码254个氨基酸。该基因核苷酸序列与中华蜜蜂Apis cerana（98.4%）高度相似,并存在15个潜在抗原表位、9个磷酸化位点和5个组氨酸磷酸酶域活性部位,属于组氨酸磷酸酶超家族,是一种二磷酸甘油酸依赖性的可溶中性稳定蛋白。荧光定量PCR结果表明,amPGAM2基因在不同品级、不同发育时期的表达差异显著（P<0.05）。工蜂卵期3日龄、幼虫期5日龄表达最高,雄蜂成虫期表达最高,蜂王幼虫期4日龄表达最高,且工蜂、雄蜂及蜂王由卵孵化成幼虫阶段和由红眼蛹羽化至成虫阶段,其表达都呈上升趋势。【结论】 本研究结果推测amPGAM2基因在卵的孵化及精卵发生过程中具有重要作用,为进一步认识意大利蜜蜂生殖发育过程中的能量代谢提供理论基础。

关键词: 意大利蜜蜂, PGAM2基因, 糖酵解, 序列特征, 荧光定量

Abstract:

[Objectives] Phosphoglycerate mutase (PGAM) plays an important role in the glycolysis and gluconeogenesis of protease. This study intends to clarify the sequence characteristics and expression patterns of the amPGAM2 gene. [Methods] The cDNA sequence of the Apis mellifera amPGAM2 gene was cloned, and its sequence characteristics and expression in different castes and developmental stages; workers, drones and queens, analyzed. [Results] Sequence analysis indicates that the full length of the cloned sequence is 976 bp, and contains a complete open reading frame encoding 254 amino acids. The nucleotide sequence of the gene is highly homologous to that of Apis cerana (98.4%). The gene has 15 potential antigen epitopes, 9 phosphorylation sites and 5 histidine phosphatase domain active sites and encodes a glycerol diphosphate-dependent soluble neutral stable protein that belongs to the histidine phosphatase superfamily. The results of RT-qPCR indicate that expression of amPGAM2 is significantly different in different castes and developmental stages. Expression in workers was highest at 3 days of age in the egg stage and at 5 days in the larval stage. The highest expression in drones and queesn was in the adult stage and in 4 day-old larvae, respectively. Expression in workers, drones and queens increased from hatching to the larval stage, and from the red-eye pupal eclosion to the adult stage. [Conclusion] The results suggest that the amPGAM2 gene plays an important role in hatching and spermatogenesis. This paper provides a theoretical basis for improving understanding of the energy metabolism during reproductive development in Apis mellifera.

Key words: Apis mellifera, PGAM2 gene, glycolysis, sequence characteristics, fluorescence quantification

欧阳霞辉, 彭帅, 徐文凯, 朱雪蕾, 郑相相, 陈红. 意大利蜜蜂amPGAM2基因的克隆、序列特征及表达分析 ^*[J]. 应用昆虫学报, 2020, 57(4): 889-897.

Xia-Hui OUYANG, Shuai PENG, Wen-Kai XU, Xue-Lei ZHU, Xiang-Xiang ZHENG, Hong CHEN. Cloning, sequence characterization and expression of the Apis mellifera amPGAM2 gene[J]. Chinese Journal of Applied Entomology, 2020, 57(4): 889-897.

图/表 9

表1 本研究所用引物信息

Table 1 Primers used in this study

引物 Primers	引物序列（5￠-3￠） Primer sequence (5￠-3￠)	引物用途 Use of primers
PGAM2-F	AATACGATAGGTGGTGGGGG	扩增引物 Amplified primers
PGAM2-R	TGAAACACATCTCTAGAAAAACATC	扩增引物 Amplified primers
q-pgam2(s)	TCCGATAAAGGTAAGATTGAAGCA	实时荧光定量PCR RT-qPCR
q-pgam2(a)	ACGTTCATTTAATCGCCATGTCTTT
qβ-actin-2F	TGCCAACACTGTCCTTTCTG
qβ-actin-2R	AGAATTGACCCACCAATCCA

图1 意大利蜜蜂amPGAM2基因PCR扩增结果 M：DL2000DNA Marker;1：阴性对照;2：amPGAM2基因PCR扩增产物。

Fig. 1 PCR amplification of amPGAM2 gene in Apis mellifera M: DL2000DNA Marker; 2: Negative control; 2: PCR product of amPGAM2 gene.

图2 采用NJ法构建的PGAM2基因系统进化树

Fig. 2 Phylogenetic tree of PGAM2 gene based on NJ method

表2 意大利蜜蜂amPGAM2蛋白理化性质

Table 2 Physicochemical properties of amPGAM2 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	C₁₃₀₆H₂₀₅₀N₃₅₀O₃₈₀S₈
等电点Isoelectric point	7.07
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)	35
不稳定指数Instability index	39.24
总平均亲水性系数Total average hydrophilicity coefficient	﹣0.505
半衰期Half life	30 h

图3 意大利蜜蜂amPGAM2蛋白三级结构

Fig. 3 Tertiary structure of amPGAM2 protein in Apis mellifera

图4 意大利蜜蜂amPGAM2蛋白B细胞抗原表位分析横轴为蛋白质的氨基酸序列,纵轴为抗原指数。位于临界值以上的峰值区间为潜在抗原表位。

Fig. 4 Analysis of B cell antigen epitopes of amPGAM2 protein in Apis mellifera The transverse axis is the amino acid sequence of protein and the longitudinal axis is the antigen index. The peak interval above the critical value is the potential antigen epitope.

表3 意大利蜜蜂amPGAM2氨基酸序列中潜在抗原表位

Table 3 Potential antigenic epitopes in the amino acid sequence of amPGAM2 in Apis mellifera

顺序 Order	氨基酸位置 Amino acid position	潜在抗原表位序列 Potential epitope sequence	得分 Score
1	13-16	ESEW	0.915
2	29-38	HLSDKGKIEA	0.860
3	43-45	KAI	0.530
4	76	Q	0.400
5	79-80	IT	0.580
6	92-93	YG	0.403
7	98-110	LNKAETAAKYGEE	1.259
8	118-131	SFDTPPPPMEPDHK	2.850
9	140-153	PRYANDPKPEEFPK	2.269
10	166	P	0.350
11	177	K	0.404
12	200-203	EMSN	0.556
13	224	N	0.353
14	226	K	0.406
15	237-241	EETVK	0.935

图5 amPGAM2基因在意大利蜜蜂不同品级、不同发育时期的表达情况 G-2：工蜂卵期2龄;G-3：工蜂卵期3日龄;G-5：工蜂幼虫期5日龄;G-7：工蜂幼虫期7日龄;G-9：工蜂幼虫期9日龄;G-11：工蜂幼虫期11日龄;G-Y：工蜂预蛹期;G-B：工蜂白眼蛹期;G-H：工蜂红眼蛹期;G-C：工蜂成虫期。X-2：雄蜂卵期2日龄;X-3：雄蜂卵期3日龄;X-4：雄蜂幼虫期4日龄;X-6：雄蜂幼虫期6日龄;X-8：雄蜂幼虫期8日龄;X-10：雄蜂幼虫期10日龄;X-12：雄蜂幼虫期12日龄;X-Y：雄蜂预蛹期;X-B：雄蜂白眼蛹期;X-H：雄蜂红眼蛹期;X-C：雄蜂成虫期。W-2：蜂王卵期2日龄;W-3：蜂王卵期3日龄;W-4：蜂王幼虫期4日龄;W-5：蜂王幼虫期5日龄;W-6：蜂王幼虫期6日龄;W-7：蜂王幼虫期7日龄;W-Y：蜂王预蛹期;W-B：蜂王白眼蛹期;W-H：蜂王红眼蛹期;W-C：蜂王成虫期。柱上标有不同字母表示经单因素方差分析差异显著（P<0.05,Duncan氏多重比较法)）。

Fig. 5 Expression of amPGAM2 gene in different grades and developmental stages of Apis mellifera G-2: Egg stage of worker bees 2 days old; G-3: Egg stage of worker bees 3 days old; G-5: Larval stage of worker bees 5 days old; G-7: Larval stage of worker bees 7 days old; G-9: Larval stage of worker bees 9 days old; G-11: Larval stage of worker bees 11 days old; G-Y: Worker bees prepupa; G-B: Worker bees white eye pupa; G-H: Worker bees red eye pupa; G-C: Worker bees adult. X-2: Egg stage of drones 2 days old; X-3: Egg stage of drones 3 days old ; X-4: Larva stage of drones 4 years old; X-6: Larva stage of drones 6 years old; X-8: Larva stage of drones 8 years old; X-10: Larva stage of drones 10 years old; X-12: Larva stage of drones 12 years old; X-Y: Drones prepupa; X-B: Drones white eye pupa; X-H: Drones red eye pupa; X-C: Drones adult. W-2: Egg stage of queen bees 2 days old; W-3: Egg stage of Queen bees 3 days old; W-4: Larva stage of queen bees 4 days old; W-5: Larva stage of queen bees 5 days old; W-6: Larva stage of queen bees 6 days old; W-7: Larva stage of queen bees 7 days old; W-Y: Queen bees pre-pupa; W-B: Queen bees white eye pupa; W-H: Queen bees red eye pupa; W-C: Queen bees adult. Histograms with different letters indicate significant difference in variance analysis of single factors (P <0.05, Duncan’s multiple comparison).

图6 amPGAM2基因在意大利蜜蜂不同品级蛹期及成虫表达情况折线图 Y：预蛹;B：白眼蛹;H：红眼蛹;C：成虫。

Fig. 6 Expression of amPGAM2 gene in pupal and adult stages of Apis mellifera Y: Prepupa; B: White-eyed pupa; H: Red-eyed pupa; C: Adult.

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