中华蜜蜂 β-GBP 基因在抵抗蜜蜂球囊菌侵染中的作用

Role of the β-1,3-glucan-binding protein gene in Apis cerana cerana against Ascosphaera apis infection

摘要:

目的 解析中华蜜蜂( Apis cerana cerana)的β-1,3-葡聚糖结合蛋白(Acβ-GBP)在响应蜜蜂球囊菌( Ascosphaera apis)侵染时的表达谱，探究干扰 Acβ-GBP对蜜蜂球囊菌侵染的幼虫死亡率和白垩病发病率的影响，为进一步的功能研究奠定基础。 方法 基于生物信息学方法分析Acβ-GBP的序列和结构特征；采用实时荧光定量逆转录PCR (real-time RT-PCR, RT-qPCR)技术调查幼虫肠道中 Acβ-GBP在球囊菌侵染后的表达特征；通过RNA干扰技术探究 Acβ-GBP对幼虫死亡率及白垩病发病率的影响。 结果 中华蜜蜂 Acβ-GBP的CDS长度为1 440 bp，编码蛋白的分子质量约为54.68 kDa，平均亲水系数为-0.22，存在典型的跨膜结构域和信号肽。进化分析表明，中华蜜蜂与小蜜蜂( Apis florea)、大蜜蜂( Apis dorsata)的β-GBP共同聚为一大支。蜜蜂球囊菌侵染后，中华蜜蜂工蜂幼虫肠道中 Acβ-GBP的表达量在1-3 dpi均显著下调( P<0.05)；干扰 Acβ-GBP后，其表达量在2 dpi和3 dpi极显著低于ds-egfp组( P<0.01)。幼虫累计死亡率和白垩病发病率均随侵染时间增加而升高，且整体死亡率极显著高于对照组( P<0.000 1)。 结论 Acβ-GBP能够响应蜜蜂球囊菌的侵染，干扰 Acβ-GBP表达显著降低蜜蜂幼虫对蜜蜂球囊菌的抵抗性。β-GBP是中华蜜蜂的重要免疫识别蛋白，在抵御真菌入侵过程中发挥重要作用。

Abstract:

Objective To analyze the expression profile of the β-glucan-binding protein (Acβ-GBP) gene of Apis cerana cerana in response to Ascosphaera apis infection and to investigate the impacts of Acβ-GBP knockdown on the larval mortality and the incidence of chalkbrood disease following A. apis infection. These findings will provide a foundation for further functional research. Methods The sequence and structural characteristics of Acβ-GBP were analyzed via bioinformatics approaches. RT-qPCR was employed to investigate the expression profiles of Acβ-GBP in the larval midgut following A. apis infection. Furthermore, RNA interference (RNAi) was utilized to explore the impacts of Acβ-GBP on the larval mortality and the incidence of chalkbrood disease. Results The CDS length of Acβ-GBP was 1 440 bp, encoding a protein with a molecular weight of 54.68 kDa and a grand average of hydropathicity value of -0.22, which contained a typical transmembrane domain and signal peptide. Phylogenetic analysis revealed that β-GBP of A. c. cerana, Apis florea and Apis dorsata clustered into a single major clade. After A. apis infection, the expression level of Acβ-GBP in the midgut of A. c. cerana worker larvae was downregulated at 1-3 days post-infection (dpi) ( P<0.05). Following RNA interference (RNAi)-mediated silencing of Acβ-GBP, its expression level was lower than that in the ds-egfp group at 2 and 3 dpi ( P<0.01). The cumulative larval mortality and the incidence of chalkbrood disease both increased over the infection time, and the overall mortality was higher than that of the control group ( P<0.000 1). Conclusion Acβ-GBP was capable of responding to A. apis infection, and knockdown of Acβ-GBP expression significantly impaired the resistance of honeybee larvae to A. apis. Collectively, β-GBP acts as an important immune recognition protein in A. c. cerana, and plays an important role in defending against fungal invasion.