棉铃虫类肌钙蛋白基因的克隆、原核表达纯化及其时空表达谱分析

Cloning,prokaryotic expression,purification and temporal-spatial expression analysis of the HaCal gene from cotton bollworm Helicoverpa armigera

为探索棉铃虫Helicoverpa armigera(Hübner)类肌钙蛋白基因HaCal的生理功能及其所编码蛋白的生物特性,采用PCR方法克隆了HaCal的开放阅读框(open reading frame,ORF)序列,借助在线软件对其进行生物信息学分析,通过原核表达技术诱导表达和纯化其编码的蛋白,并通过实时荧光定量PCR技术分析其在棉铃虫不同发育龄期和组织中的表达量。结果显示,HaCal的ORF序列长度为564 bp,编码187个氨基酸,理论分子量为20.52 kD,理论等电点为7.64。其保守结构域与Calponin家族一致,但不含跨膜区和信号肽,系亲水性蛋白。原核表达结果显示,融合蛋白大小与理论值一致,纯化获得了纯度较高的目的蛋白。HaCal在棉铃虫幼虫不同发育阶段和不同组织中均有表达,在6龄幼虫体内表达量最高,是1龄幼虫的2.34倍;在5龄幼虫中肠中表达量最高,是头部的257.14倍。表明棉铃虫类肌钙蛋白基因HaCal可能与棉铃虫的生长发育过程密切相关。

In order to explore the physiological function of HaCal（calponin） and the biological characteristics of its protein, HaCal open reading frame（ORF） sequences were cloned by PCR. Bioinformatics analysis of HaCal ORF sequence was carried out by means of on-line software. The expression of HaCal gene was induced by prokaryotic expression technology. The expressed protein was purified, and quantified by real-time fluorescence quantitative polymerase chain reaction in different developmental stages and tissues of cotton bollworm Helicoverpa armigera（Hübner）. The results showed that the sequence length of HaCal ORF was 564 bp, encoding 187 amino acids, and its theoretical molecular weight was 20.52 kD with a theoretical isoelectric point of 7.64. The conserved domains were consistent with that of the Calponin family, but contained no transmembrane domain and signal peptide, indicating that it was a hydrophilic protein. The results of prokaryotic expression showed that the size of the fusion protein was consistent with the theoretical value, and the target protein with high purity was obtained. HaCal was expressed during different developmental stages and in different tissues of H. armigera larvae, and the expression of HaCal in the 6 th-instar larvae was 2.34 times higher than that of the1 st-instar larvae. In different tissues of the larvae, the expression of HaCal was the highest in the midgut, which was 257.14 times of that in the head in the 5 st-instar larvae. The HaCal gene of H. armigera might be closely related to the growth and development of H. armigera.