桑黄鲨烯环氧酶基因原核表达及过表达载体的构建  

Construction of prokaryotic expression and over-expression vectors of squalene epoxidase gene from Sanghuangporus baumii

作　　者：孙婷婷 孙煦茵 王旭彤[2] 王世新[2] 刘增才 邹莉[2] SUN Ting-ting;SUN Xu-yin;WANG Xu-tong;WANG Shi-xin;LIU Zeng-cai;ZOU Li(Department of Food Engineering, Harbin University, Harbin 150086, China;College of Forestry, Northeast Forestry University, Harbin 150040, China)

机构地区：[1]哈尔滨学院食品工程学院,黑龙江哈尔滨150086 [2]东北林业大学林学院,黑龙江哈尔滨150040

出　　处：《中草药》2018年第11期2632-2639,共8页Chinese Traditional and Herbal Drugs

基　　金：中央高校基本科研业务费专项资金资助(2572017CF01);哈尔滨学院青年博士科研启动基金项目(HUDF2018105)

摘　　要：目的构建桑黄三萜生物合成途径中的关键酶——鲨烯环氧酶(squalene epoxidase,SE)基因的原核表达和过表达载体。方法将克隆到的桑黄SE基因构建到原核表达载体p ET-32a(+)上,转化大肠杆菌BL21(DE3),使用异丙基硫代-β-D-呋喃半乳糖苷(IPTG)诱导表达2~10 h后,通过SDS-PAGE进行检测。根据Gen Bank中提交的香菇3-磷酸甘油醛脱氢酶基因(gpd)启动子序列设计引物,利用PCR技术克隆获得香菇gpd启动子片段,以植物双元表达载体p CAMBIA1301为基础载体,通过酶切、连接等方法将其中的35 S启动子替换为香菇gpd启动子,再将SE基因编码区序列构建到gpd启动子下游,构建桑黄SE基因的过表达载体。结果成功构建了SE基因的原核表达载体p ET-32a-SE,SDS-PAGE结果显示该蛋白的大小约为55 000,与预测的蛋白相对分子质量基本一致。PCR检测、酶切鉴定和测序分析可知,成功构建了适宜食用菌遗传转化的中间载体p CAMBIA1301-gpd-gpd及SE基因的过表达载体p CAMBIA1301-gpd-gpd-SE。结论为后期研究SE基因在桑黄三萜生物合成方面所发挥的功能奠定基础。Objective To obtain prokaryotic expression and over-expression vectors of squalene epoxidase（SE） gene from Sanghuangporus baumii. Methods The entire protein-coding c DNA of SE was cloned into the expression vector p ET-32 a（＋）. Then the recombinant plasmid was transformed into Escherichia coli BL21（DE3） cells. SDS-PAGE was used to investigate the situation of expression after IPTG induction for 2—10 h. Additionally, primers were designed according to the gpd promoter sequence of Lentinula edodes in Gen Bank, and the gpd promoter fragment was obtained by PCR. Subsequently, the plant binary expression vector p CAMBIA1301 was selected as the basic vector, and then the 35 S promoter replaced with L. edodes gpd promoter through enzyme digestion and connection. Finally, the coding region of SE was cloned to the downstream of the gpd promoter to construct over-expression vectors. Results The prokaryotic expression vector p ET-32 a-SE was successfully obtained. SDS-PAGE results showed a significant protein band was found in the vicinity of the relative molecular weight of approximately 55 000, consistent with molecular weight of the predicted protein. Moreover, the over-expression vector p CAMBIA1301-gpd-gpd-SE was constructed successfully through different detection ways. Conclusion These results lay the foundation for the further study of SE in triterpenoid biosynthesis pathway of S. baumii.

关 键 词：桑黄 鲨烯环氧酶 原核表达 载体构建 PCR 

分 类 号：R282.12[医药卫生—中药学]