藏药川西獐牙菜中SmMK基因的克隆及生物信息分析  

Cloning and bioinformatics analysis of SmMK gene in Swertia mussotii

作　　者：李晓雪[1] 向蓓蓓[2] 孙继奇 白艳玲[1] 朱晔荣[1] 马琳[2] 冯林林 王勇[1] LI Xiao-xue;XIANG Bei-bei;SUN Ji-qi;BAI Yan-ling;ZHU Ye-rong;MA Lin;FENG Lin-lin;WANG Yong(College of Life Science,Nankai University,Tianjin 300071,China;School of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin 300193,China)

机构地区：[1]南开大学生命科学学院,天津300071 [2]天津中医药大学中药学院,天津300193

出　　处：《中草药》2019年第13期3169-3177,共9页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金资助项目(81303303);天津市高等学校科技发展基金计划项目(20130203)

摘　　要：目的了解川西獐牙菜Swertia mussotii Franch甲羟戊酸途径中的关键酶甲羟戊酸激酶基因(MK)的功能,并进一步推进川西獐牙菜中的甲羟戊酸途径的研究。方法根据川西獐牙菜的转录组信息,获得了川西獐牙菜MK(SmMK)基因的全长cDNA序列,并且设计了特异性引物,利用RT-PCR对该基因进行了克隆;利用生物信息学方法,对SmMK基因的序列进行分析;构建原核表达载体MBP-SmMK,转入大肠杆菌Rosetta(DE3)中,然后对Sm MK基因进行了原核表达,并对其进行了纯化。结果SmMK基因cDNA全长为1 164 bp,共编码387个氨基酸。SmMK与其他植物中的MK具有较高相似性。对其信号肽、跨膜区域、蛋白定位及二级结构和三维构象进行了预测分析。SDS-PAGE检测表明所纯化蛋白与预期蛋白的大小相一致,为40970。结论为进一步研究川西獐牙菜中MK的功能奠定了基础,并为进一步研究川西獐牙菜中的甲羟戊酸途径,提高川西獐牙菜中类异戊二烯化合物的产量提供了依据。Objective In order to identify the function of the mevalonate kinase(MK) which is a key enzyme of the mevalonate pathway(MVA) in Swertia mussotii, and to improve the study of MVA in S. mussotii. Methods According to the SmMK gene sequence of transcriptome of S. mussotii, the specific primers were designed, the cDNA complete sequences was obtained by RT-PCR and the sequence was analyzed using bioinformatics. Prokaryotic expression vector MBP-SmMK was constructed and transformed into Escherichia coli Rosetta(DE3) for expression. Results The results showed that SmMK cDNA complete sequences had a length of1 164 bp encoding 387 amino acid residues. The SmMK protein shared high identity with other MK proteins of plants. And the protein signal peptide, transmembrane region, location, secondary, and tertiary structures were analyzed and forecasted. The SDS-PAGE results showed that the expressed proteins were consistent with the anticipated size, which was 40 970. Conclusion This work will provide a foundation for research the SmMK protein functional and study MCA in S. mussotii. At the same time, it will supply the basis to improve the production of the isoprenoids.

关 键 词：川西獐牙菜 甲羟戊酸激酶 基因克隆 生物信息学分析 原核表达 

分 类 号：R282.12[医药卫生—中药学]