摘要
[目的]研究茶尺蠖(Ectropis oblique) Eo GST534原核表达,及其在溴氰菊酯处理下的体内表达规律。[方法]进行Eo GST534生物信息学分析,将重组质粒p ET-32a-Eo GST534转化大肠杆菌BL21中,诱导表达并纯化,将重组蛋白进行酶活测定。使用不同浓度的溴氰菊酯处理茶尺蠖,使用qRT-PCR检测目的基因表达量。[结果]成功表达目的蛋白,大小约为40 k Da。其最适温度为50℃,最适p H为10. 5时酶活为7. 32 U/mg。使用溴氰菊酯处理不同龄期的幼虫。在IC50浓度溴氰菊酯处理不同龄期幼虫12 h后,2、3、4龄幼虫Eo GST534表达量显著降低。而3、4龄幼虫Eo GST534表达量在处理后24 h后升高,其表达量变化分别约为20倍和26倍。在IC25浓度溴氰菊酯处理12 h后,Eo GST534在2龄及3龄幼虫中表达量均显著降低。而在处理24 h后,4龄幼虫Eo GST534表达量显著增加约10倍。[结论]原核表达了重组Eo GST534,并测定了其最适条件酶活为7. 32 U/mg。初步研究了其在溴氰菊酯处理的茶尺蠖体内Eo GST534表达量变化规律。
[Objective]The research was to study the prokaryotic expression of Ectropis oblique EoGST534 and its expression in vivo under deltamethrin treatment.[Method]EoGST534 bioinformatics analysis was carried out,and the recombinant plasmid pET-32a-EoGST534 was transformed into E.coli BL21 to induce expression and purification,and the recombinant protein was subjected to enzyme activity assay.The Ectropis oblique were treated with different concentrations of deltamethrin,and the expression of the target gene was detected by qRT-PCR.[Result]The target protein was successfully expressed and the size was about 40 kDa.The enzyme activity is 7.32 U/mg when at the optimum pH of 10.5 and optimum temperature of 50℃.larvae of different ages was treat by different concentration of deltamethrin.The expression of EoGST534 in 2,3,and 4 ages larvaes were significantly decreased after treatment of different age larvae with IC50 concentration of deltamethrin for 12 hours.The expression level of EoGST534 in 3rd and 4th age larvae increased after 24hours,and the expression changes were about 20 times and 26 times,respectively.The expression of EoGST534 was significantly decreased in the 2nd and 3rd age larvae after 12 h treatment with IC25 concentration of deltamethrin.After 24 hours of treatment,the expression level of EoGST534 of 4th instar larvae increased significantly by about 10 times.[Conclusion]EoGST534 was expressed in prokaryotic cells,and its optimal condition was determined to be 7.32 U/mg.The changes of EoGST534 expression in the deltamethrin-treated Ectropis oblique were studied.
作者
周雨晴
毛新芳
刘忠渊
ZHOU Yu-qing;MAO Xin-fang;LIU Zhong-yuan(Department of Life Science and Technology,Xinjiang University,Urumqi 830046,China;Sichuan University of Science&Engineering,Zigong 643000,China)
出处
《生物技术》
CAS
2019年第6期525-532,共8页
Biotechnology
基金
四川省应用基础研究项目(2019YJ0459)
四川轻化工大学人才引进项目(2017RCL63)
