棉铃虫不同品系Gqα基因的比较及其原核表达与纯化

Gqα gene in different Helicoverpa armigera strains and its prokaryotic expression

为了阐明Gqα的DNA序列情况,分别以cDNA和总DNA为模板,对Gqα进行PCR扩增,结果发现,Gqα在DNA水平上没有内含子。为了了解不同棉铃虫品系Gqα的变异情况,以实验室5个棉铃虫品系的中肠总DNA为模板,使用Pyrobest高保真酶对该基因进行扩增,测序后翻译成氨基酸序列发现,田间的耐药性品系只有2个氨基酸的变异,即:第163位的非极性A(丙氨酸)变成了极性T(苏氨酸);第353位的非极性V(缬氨酸)变成了碱性R(精氨酸);实验室内毒蛋白品系只有一个氨基酸的变异,第353位的V变成了R;其他2种实验室品系没有任何氨基酸的变异。个别氨基酸的变异可能与棉铃虫对Bt毒素产生抗性相关。将Gqα编码区克隆到表达载体pET21b中,表达出带有6个组氨酸标签的重组蛋白并对其进行了纯化,为进一步研究其功能奠定了基础。

It was found that Gqα in Helicoverpa armigera contained no introns based on PCR amplification. Gqα was amplified with Hi-Fidelity DNA polymerase and total DNA from midgut of 5H. armigera strains, namely sensitive strain, resistant strain in the laboratory, 5.0 strain in the laboratory, toxic protein strain in the laboratory and toxin-tolerant strain in the field. Compared to the sensitive strain, the toxin-tolerant strain in the field had a Gqα with A at the position 163 was replaced by T, and V at the position 353 with R; the toxic protein strain had only one amino acid replaced （ V to R at the position 353） ; the other two strains had no mutations at all. The mutation of some amino acids might be responsible for the resistance of H. armigera to Bt toxin. The coding region of Gqα was cloned to pET21b, and the recombinant fusion protein with 6-His tag was successfully expressed, and then purified, which is helpful for further investigation of Gqa function.