扶桑绵粉蚧钙调蛋白基因的原核表达与表达谱分析

Prokaryotic expression and expression profiling of calmodulin genes in mealybug Phenacoccus solenopsis Tinsley(Hemiptera:Pseudococcidae)

【目的】探究我国重要入侵生物扶桑绵粉蚧(Phenacoccus solenopsis Tinsley)钙调蛋白基因(PsCam)在各虫态中的表达差异及PsCam蛋白在体外的异源表达,为进一步揭示PsCam的生理功能提供参考。【方法】克隆PsCam基因的ORF,推导其编码的氨基酸序列,采用生物信息学方法,分析扶桑绵粉蚧钙调蛋白的结构。构建PsCam原核表达载体,将其转化到大肠杆菌中进行诱导表达,并对产物进行纯化。采用实时荧光PCR方法,分析该基因在扶桑绵粉蚧各个虫态中的相对表达量。【结果】扶桑绵粉蚧钙调蛋白基因的氨基酸序列分析结果显示,该蛋白没有信号肽,具有2个EF-hand钙结合结构域,有13个Ca2+结合位点。成功构建了原核表达载体pET28a-PsCam,经诱导表达后获得了重组的Cam蛋白,目的蛋白的分子质量约为17ku。PsCam mRNA在不同虫态的扶桑绵粉蚧中都有表达,在成熟雌虫中的表达量最低,在1龄幼虫中的表达量最高,是成熟雌虫的7.1倍。【结论】在原核表达系统中诱导表达了重组PsCam蛋白;PsCam基因在扶桑绵粉蚧不同虫态中差异表达。

【Objective】The research investigated the expression of calmodulin gene in Phenacoccus solenopsis （PsCam） at different developmental stages and its heterologous expression in vitro to improve the further exploration of its physiological function.【Method】The ORF of PsCam was cloned,the amino acid sequence was derived,and its structure was analyzed using bioinformatics methods.The prokaryotic expression vector was constructed before being transformed into E.coli for expression and purification.Its relative expression levels at different instars were analyzed using real-time PCR.【Result】The amino acid sequence indicated that there were two EF-hand domains and 13 Ca2＋ binding sites but no signal peptide in the protein of PsCam.The prokaryotic expression vector of pET28aPsCam was successfully built.The relative molecular mass of the obtained protein after induced expression was approximately 17 ku.Expression of PsCam mRNA was observed in different instars of P.solenopsis.The expression in mature females was the lowest,while the expression in one year old instar larvae was the highest,which was 7.1 times of that in mature females.【Conclusion】P.solenopsis calmodulin protein was successfully expressed and the expression levels in different instars were different.