川西獐牙菜SmDXS2基因的克隆及表达分析  

Cloning and expression of SmDXS2 gene in Swertia mussotii

作　　者：李文静 向蓓蓓[2] 孙艳香[1] 侯晓强[1] 韩美玲 李晓雪[3] 王勇[3] 果硕 LI Wen-jing;XIANG Bei-bei;SUN Yan-xiang;HOU Xiao-qiang;HAN Mei-ling;LI Xiao-xue;WANG Yong;GUO Shuo(College of Life Science,hingfang Teachers University,Langfang 065000,China;School of Chinese Materia Medica,Tianjin University of Traditional Chinese Medicine,Tianjin 300193,China;College of Life Science,Nankai University,Tianjin 300071,China)

机构地区：[1]廊坊师范学院生命科学学院,河北廊坊065000 [2]天津中医药大学中药学院,天津300193 [3]南开大学生命科学学院,天津300071

出　　处：《中国中药杂志》2019年第5期935-941,共7页China Journal of Chinese Materia Medica

基　　金：河北省高等学校科学技术研究青年基金项目(QN2018017);天津市自然科学基金项目(18JCQNJC14000)

摘　　要：1-脱氧-D-木酮糖-5-磷酸合成酶(1-deoxy-D-xylulose-5-phosphate synthase,DXS)是2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径的第一个关键酶。该研究根据川西獐牙菜Swertia mussotii转录组数据,克隆获得其DXS2基因(Gen Bank注册号MH535905),命名为Sm DXS2。生物信息学分析表明Sm DXS2基因无内含子,其开放阅读框全长2 145 bp,编码714个氨基酸,分别属于20种氨基酸,其中丙氨酸、甘氨酸和色氨酸含量最高;相对分子质量为76. 91 k Da,理论等电点为6. 50,属亲水性蛋白;二级结构中α-螺旋占36. 83%,延伸链占9. 38%,loop占53. 78%;序列内部无信号肽,没有跨膜区,为非分泌型蛋白;序列中含有TPP结合位点、嘧啶结合结构域和C端的转酮酶结构域;系统发育分析结果显示Sm DXS2与滇龙胆DXS2共聚于一个分支。Sm DXS2基因在大肠杆菌中表达的重组蛋白相对分子质量约为94 k Da,与预测蛋白大小一致。该研究为进一步探讨该基因的功能和利用基因工程手段提高川西獐牙菜中环烯醚萜类化合物产量提供了理论依据。1-deoxy-D-xylulose-5-phosphate synthase2(DXS2) is the first key enzyme of the MEP pathway,which plays an important role in terpene biosynthesis of plants. According to the data of Swertia mussotii transcriptome, DXS2 gene(Gen Bank number MH535905) was cloned and named as Sm DXS2. The bioinformatics results showed that Sm DXS2 has no intron,with a 2 145 bp open reading frame encoding a polypeptide of 714 amino acids. They are belonging to 20 kinds of amino acids,and the most abundant amino acids include Ala,Gly and Trp. The predicted protein molecular weight was 76. 91 k Da and its theoretical isoelectric point(p I) was6. 5,which belonging to a hydrophilic protein.α-Helix and loop were the major motifs of predicted secondary structure of DXS2. The three function domains are TPP_superfamily,Transket_pyr_ superfamily and Transketolase_C superfamily,respectively. The Sm DXS2 protein shared high identity with other DXS2 proteins of plants. Phylogenetic analysis showed that Sm DXS2 protein is grouped with the gentian DXS2 protein. The recombinant protein of Sm DXS2 gene in Escherichia coli was approximately 92. 00 k Da(containing sumo-His tag protein 13 k Da),which was consistent with the anticipated size.This work will provide a foundation for further functional research of Sm DXS2 protein and increasing the product of iridoid compound by genetic engineering in S. mussotii.

关 键 词：川西獐牙菜 1-脱氧-D-木酮糖-5-磷酸合成酶2 基因克隆 生物信息学分析 原核表达 

分 类 号：S567.2[农业科学—中草药栽培]