Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (12): 2461-2471.doi: 10.3864/j.issn.0578-1752.2022.12.016


Molecular Cloning and Expression Pattern Analysis of NPC2 Gene Family of Apis cerana cerana

ZHANG Li(),ZHANG Nan,JIANG HuQiang,WU Fan,LI HongLiang()   

  1. College of Life Sciences, China Jiliang University/Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, Hangzhou 310018
  • Received:2021-12-19 Accepted:2022-01-21 Online:2022-06-16 Published:2022-06-23
  • Contact: HongLiang LI;


【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

Table 1

List of the primer information of the NPC2 genes"

Primer name
Primer sequence (5′-3′)
Product length (bp)
Tm (℃)
用于基因克隆 For gene cloning
用于实时荧光定量PCR For qRT-PCR
酶切位点用下划线表示The underlines represent the restriction sites

Table 2

Sequence characteristics and physicochemical properties of AcNPC2 gene family"

GenBank accession number
ORF full length (bp)
Length of encoding protein (aa)
Molecular weight
Isoelectric point (pI)
Similarity to the nucleotide sequence of A. m. ligustica (%, E value)
AcNPC2a KJ633823.1 447 148 16.12 7.98 94.59%, 1e-98
AcNPC2b OL741688 480 159 18.53 7.57 96.86%, 2e-113
AcNPC2c OL741689 459 152 16.96 6.56 98.03%, 8e-103
AcNPC2d OL741690 465 154 16.78 6.34 99.35%, 2e-108

Fig. 1

Multiple sequences alignment between AcNPC2s and homologous protein sequences The N-terminal box part is the predicted signal peptide sequence; Asterisks mean the conserved cysteine; The different amino acids between AcNPC2s and AmNPC2s were shown by bold fonts and underline; HomNPC2, BovNPC2 and CjapNPC2 belong to Homo sapiens NPC2, bovine NPC2 and Camponotus japonicus NPC2, respectively"

Fig. 2

Phylogenetic tree based on AcNPC2 gene family"

Table 3

Three-dimensional model scoring of AcNPC2 proteins"

蛋白Protein 描述Description Template Seq Identity GMQE QMEAN
AcNPC2a 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

Fig. 3

Three-dimensional structure prediction of AcNPC2 gene family protein A-H are the best predicted three-dimensional models of AcNPC2 proteins and their corresponding surface models, respectively, red indicates disulfide bonds, the yellow parts are the β-fold structures, and the arrow indicates the amino acid sequence from N-terminal to C-terminal"

Fig. 4

Expression profile of AcNPC2 gene family in tissues of worker, and in eggs, larvae, and pupae"

Fig. 5

Expression of AcNPC2 gene family in the antenna of foragers at 12℃ and 25℃"

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