中华蜜蜂NPC2基因家族克隆及表达模式分析

doi:10.3864/j.issn.0578-1752.2022.12.016

摘要/Abstract

摘要：

【背景】作为我国本土重要的资源昆虫,中华蜜蜂（Apis cerana cerana,简称中蜂）对我国初冬季低温开花植物的传粉具有重要的生态价值,其传粉习性与嗅觉系统密切相关。前期对中蜂采集蜂高、低温处理后的触角转录组数据分析发现,与昆虫嗅觉相关的尼曼匹克C2型蛋白（Niemann-Pick type C2 protein,NPC2）基因家族在低温时表达量上升。【目的】以中蜂NPC2家族基因为研究对象,克隆并分析其结构特征和表达谱以及高、低温处理下表达量的差异,为深入研究中蜂NPC2基因家族在中蜂低温适应的嗅觉感受功能提供参考。【方法】基于中蜂高、低温转录组测序结果,利用RT-PCR克隆获得中蜂NPC2基因ORF序列,进行系统进化树分析和三维结构预测,然后通过实时荧光定量PCR分析中蜂NPC2基因在不同发育时期、组织的时空表达谱,以及高、低温时的表达量变化。【结果】获得4个中蜂NPC2基因——AcNPC2a、AcNPC2b、AcNPC2c、AcNPC2d的ORF全长,分别为447、480、459和465 bp,编码148、159、152和154个氨基酸,预测蛋白分子量为16.12—18.53 kD,等电点分别为7.98、7.57、6.56和6.34。进化树分析显示AcNPC2与意大利蜜蜂的NPC2同源序列亲缘关系最近,与其他膜翅目昆虫NPC2也有一定相似性。实时荧光定量PCR结果显示,AcNPC2a在新出房蜂的腹部表达量最高,其次是在哺育蜂的腹部以及幼虫期;AcNPC2b在新出房蜂的胸部表达最高,在采集蜂的头部、胸部和后足也有表达;AcNPC2c在哺育蜂和采集蜂的触角中呈高丰度表达;AcNPC2d在采集蜂的头部表达量最高。经低温处理后,4个AcNPC2基因在采集蜂触角中的表达量均有所上升,但差异不显著。【结论】 AcNPC2具有NPC2蛋白的保守结构,其基因家族成员在中蜂的时空表达谱中呈现多样性,其中AcNPC2c在触角中呈高丰度表达,表明其与中蜂嗅觉感受功能关系密切。AcNPC2基因家族在低温时采集蜂触角中表达量均有所上升,表明该基因家族有可能参与中蜂的低温适应性,或与初冬季访花行为有关。

关键词: 中华蜜蜂, 尼曼匹克C2型蛋白（NPC2）, 基因克隆, 时空表达谱

Abstract:

【Background】As an important native resource insect, the Chinese honeybee (Apis cerana cerana) plays important ecological roles in pollinating the plants that bloom at low temperatures in early winter in China, and the pollination behavior of A. c. cerana is closely related to its olfactory system. According to the analysis of antenna transcriptome data collected from foragers treated at high and low temperatures, it was found that the Niemann-Pick type C2 protein (NPC2) gene family related to insect olfaction was up-regulated expression at low temperatures.【Objective】Therefore, this study aims on the A. c. cerana NPC2 family genes, including cloning and analysis of their structural characteristics and expression profiles. Moreover, the NPC2 family gene expression under high and low temperatures was also studied. It will provide an evidence of the AcNPC2 gene family in the low-temperature adaptation involved in the chemosensory and olfactory function of A. c. cerana.【Method】Based on the results of high and low temperatures transcriptome sequencing of A. c. cerana, the ORF sequence of AcNPC2 genes was cloned by RT-PCR, and phylogenetic tree analysis and three-dimensional structure prediction were performed. Then, the spatio-temporal expression profile of AcNPC2 genes in different developmental stages and different tissues, as well as the amount of expression at high and low temperatures of A. c. cerana were analyzed by qRT-PCR.【Result】The full length ORFs of four NPC2 genes of A. c. cerana (AcNPC2a, AcNPC2b, AcNPC2c, and AcNPC2d) were obtained as 447, 480, 459, and 465 bp, respectively, encoding 148, 159, 152, and 154 amino acids. The predicted protein molecular weight is 16.12-18.53 kD, and the isoelectric points are 7.98, 7.57, 6.56, and 6.34, respectively. Phylogenetic tree analysis showed that AcNPC2 sequences were most close to the NPC2 homologous sequence of Apis mellifera ligustica. qRT-PCR results showed that the expression level of AcNPC2a was the highest in the abdomen of the newborns, followed by the abdomen of the nurses and the larval stage. The expression of AcNPC2b was the highest in the thorax of the newborn bees, followed by the head, thorax and metapodium of the foragers. AcNPC2c was notably expressed in high abundance in the antennae of the nurses and the foragers. AcNPC2d had the highest expression in the head of the foragers. After low temperature treatment, the expression levels of all AcNPC2 genes in the forager antennae increased, but there was no significant difference.【Conclusion】AcNPC2 has the conserved structure of NPC2 protein family, and its members show diversity in the spatio-temporal expression profile of A. c. cerana. Among them, AcNPC2c is highly expressed in the antennae, indicating that it is closely related to the olfactory function of A. c. cerana. The expression of the whole AcNPC2 family genes increased in the antennae of the foragers at lower temperature, indicating that these genes might be involved in the low-temperature adaptability of A. c. cerana and pollination behavior in early winter.

Key words: Apis cerana cerana, NPC2, gene cloning, spatio-temporal expression profile

张莉,张楠,江虎强,吴帆,李红亮. 中华蜜蜂NPC2基因家族克隆及表达模式分析[J]. 中国农业科学, 2022, 55(12): 2461-2471.

ZHANG Li,ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang. Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana[J]. Scientia Agricultura Sinica, 2022, 55(12): 2461-2471.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2022.12.016

https://www.chinaagrisci.com/CN/Y2022/V55/I12/2461

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引物名称 Primer name		引物序列 Primer sequence (5′-3′)	产物长度 Product length (bp)	退火温度 Tm (℃)
用于基因克隆 For gene cloning
NPC2a	NPC2e-Fa	CCGAATTCATGGCAATCCTCACCTATGTT	463	55
NPC2a	NPC2e-Ra	GGCTCGAGTTAGTCGACAACTTTAGCTGG	463	55
NPC2b	NPC2e-Fb	CCGAATTCATGAATCGAAACGGTATGTTC	496	58
NPC2b	NPC2e-Rb	GGCTCGAGTTAGTCGACAACTTTAGCTGG	496	58
NPC2c	NPC2e-Fc	CCGAATTCATGTATCGAATGATTGCTGTAA	475	60
NPC2c	NPC2e-Rc	GGCTCGAGCTATTTAATTCGAGCCGGAAT	475	60
NPC2d	NPC2e-Fd	CCGAATTCATGAGGGGAACCATTCTCGTT	481	61
NPC2d	NPC2e-Rd	GGCTCGAGTCAATTGGTGATCTTCGCTG	481	61
用于实时荧光定量PCR For qRT-PCR
NPC2a	NPC2q-Fa	ACGTTGACTTTAAGGCTGTTGC	129	51
NPC2a	NPC2q-Ra	ACTGTCCGCTCACCAGATTCTT	129	51
NPC2b	NPC2q-Fb	TCCATTATACGCCTGCCTTCTCCT	116	53
NPC2b	NPC2q-Rb	GGAAGTTGACAAGCAAGCATCGG	116	53
NPC2c	NPC2q-Fc	GATGCTTGCCAAACTCCTGATTCT	116	55
NPC2c	NPC2q-Rc	ACAGTAACACTCACCTTAGGATACG	116	55
NPC2d	NPC2q-Fd	TTCGTAGCTCTGCTCGTAGTTGTCT	138	54
NPC2d	NPC2q-Rd	CGCCTTCGTCCTTCGTTTCAGTT	138	54
内参 Reference	β-actinq-F	TGCCAACACTGTCCTTTCTGGA	96	53
内参 Reference	β-actinq-R	TTCATGGTGGATGGTGCTAGGG	96	53
酶切位点用下划线表示The underlines represent the restriction sites

蛋白Protein	描述Description	Template	Seq Identity	GMQE	QMEAN
AcNPC2a	人Homo sapiens	5kwy.1.C	32.74%	0.68	-1.25
AcNPC2b	牛Bovine	2hka.1.A	28.57%	0.55	-2.20
AcNPC2c	日本弓背蚁C. japonicus	3wea.1.A	39.53%	0.77	-0.15
AcNPC2d	日本弓背蚁C. japonicus	3wea.2.A	33.06%	0.63	-2.96