MED烟粉虱钙结合蛋白的鉴定及表达分析

Identification and expression of the calcium-binding protein in MED Bemisia tabaci(Gennadius)

【目的】钙结合蛋白作为钙信号传导途径的组分,通过与钙离子结合实现其生物学功能,调控细胞信号传导和细胞生命周期等过程,且钙结合蛋白可以通过与植物中钙离子结合降低钙离子浓度来保持昆虫的持续摄取。为明确钙结合蛋白在MED (Mediterranean)烟粉虱Bemisia tabaci体内的时空表达模式,烟粉虱取食后钙结合蛋白的表达特征及取食不同寄主的烟粉虱体内钙结合蛋白的表达量变化情况。【方法】利用RT-PCR和基因克隆得到烟粉虱钙结合蛋白cDNA序列,并通过实时荧光定量PCR(qRT-PCR)技术分析其在烟粉虱不同组织、不同龄期、不同寄主以及烟粉虱饥饿诱导取食后不同时间点的表达模式。【结果】生物信息学分析结果显示:烟粉虱钙结合蛋白基因的开放阅读框为669bp,编码一个由222个氨基酸组成的蛋白,其N端包含一段长为22个AA的信号肽序列,C末端具有钙离子结合位点。系统进化分析表明:烟粉虱钙结合蛋白与半翅目昆虫关系最近,分属同支。荧光定量PCR结果显示:钙结合蛋白在烟粉虱头部的表达量显著高于胸部、腹部、足和翅的表达量;钙结合蛋白在烟粉虱整个发育阶段均有表达,4龄若虫的表达量最高,卵期表达量最低,1-2龄、3龄、4龄和成虫期的表达量分别为卵期表达量的4.38倍、5.47倍、16.76倍和5.03倍;钙结合蛋白在取食棉花、辣椒和番茄3种不同寄主的烟粉虱中的表达量无显著性差异;饥饿诱导取食后1 h的烟粉虱成虫钙结合蛋白的表达量显著高于取食0 h,为取食0h的2.12倍。【结论】本研究克隆了烟粉虱钙结合蛋白基因并对其在烟粉虱中的表达模式进行分析,为进一步明确烟粉虱钙结合蛋白功能研究奠定基础。

[Objectives]To clarify the temporal and spatial expression of calcium-binding protein in MED(Mediterranean)Bemisia tabaci after feeding on different hosts.A component of the calcium signaling pathway,the main function of the calcium-binding protein is to bind to calcium ions,regulating cell signaling and cell life cycle processes.Calcium-binding proteins can reduce the calcium ion concentration by binding to calcium ions in plants,thereby maintaining a continuous intake by insects.[Methods]The cDNA sequence of the B.tabaci calcium binding protein was amplified using the reverse transcription polymerase chain reaction(RT-PCR)and gene cloning,and its expression in all developmental tissues and stages,hosts,and after feeding on different plants,analyzed using Real-time quantitative PCR.[Results]Bioinformatics analysis indicates that the open reading frame of the B.tabaci calcium-binding protein gene is 669 bp,encoding a protein comprised of 222 amino acids.The N-terminus contains a signal peptide sequence of 22 AA and the C-terminal has a Ca2+binding site.Phylogenetic analysis shows that the B.tabaci calcium-binding protein is closely related to that of other Hemipteran insects,and belongs to the same branch.The results of real-time PCR show that the expression of the calcium-binding protein in the head of B.tabaci was significantly higher than in the thorax,abdomen,leg and wing.The calcium-binding protein was expressed in every developmental stage of B.tabaci;expression was highest in fourth-instar nymphs and lowest in eggs.Expression levels in first,second,third and fourth instars,and in adults,were 4.38 times,5.47 times,16.76 times and 5.03 times,higher than in the egg stage,respectively.Feeding B.tabaci on pepper,tomato and cotton had no significant effect on the expression of the calcium-binding protein.The expression of the calcium-binding protein in adults that were allowed to fed 1 h after starvation was significantly(2.12 times)higher than in those fed 0 h after starvation.[Conclusion]The calcium-binding protein gene of B.tabaci can be successfully cloned and its expression pattern analyzed.The results of this study provide a foundation for further study of the function of this gene in the development of B.tabaci.