桑黄HMGS基因克隆、分子特性及差异表达分析  

Cloning, molecular properties and differential expression analysis of HMGS gene from Sanghuangporus baumii

作　　者：孙婷婷 刘增才 王世新[2] 王旭彤[2] 邹莉[2] SUN Ting-ting;LIU Zeng-cai;WANG Shi-xin;WANG Xu-tong;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》2019年第23期5823-5829,共7页Chinese Traditional and Herbal Drugs

基　　金：哈尔滨学院青年博士科研启动基金项目（HUDF2018105）

摘　　要：目的对参与桑黄三萜合成途径的关键酶羟甲基戊二酰辅酶A合酶(3-hydroxy-3-methylglutaryCoAsynthase,HMGS)进行基因全长克隆、分子特性及差异表达分析研究。方法采用RACE技术克隆HMGS基因全长,并对其序列进行生物信息学分析;构建桑黄HMGS基因到表达载体pET-32a(+)上,转化大肠杆菌BL21(DE3),IPTG诱导表达后SDS-PAGE检测;用荧光定量PCR的方法分析其不同发育阶段表达特性。结果HMGS基因cDNA全长为1930 bp,包含1个1458 bp的完整开放阅读框,编码485个氨基酸。该基因编码蛋白的相对分子质量约为52750,理论等电点是5.60,为亲水性蛋白,不具有跨膜结构,不含信号肽序列;系统发育分析表明,桑黄HMGS与地中海拟层孔菌Fomitiporia mediterranea中的HMGS同源性最高,属于一个分支。SDS-PAGE结果显示该蛋白的大小与预测的蛋白相对分子质量基本一致。HMGS基因的转录水平在桑黄不同发育阶段呈动态变化,该基因在菌丝体阶段的转录水平要高于原基及子实体阶段的转录水平。HMGS基因的最高转录水平出现在第14天,为对照第5天的2.33倍。结论HMGS基因的分子鉴定为进一步解析该基因在桑黄三萜类物质合成代谢途径中的分子调控作用奠定基础。Objective To clone and characterize a 3-hydroxy-3-methylglutaryl-CoA synthase(HMGS)gene which involved in the triterpenoid biosynthesis pathway in Sanghuangporus baumii.Methods The HMGS gene cDNA full-length sequence was cloned by RACE technology.Characteristics including the physicochemical properties and conserved domain of the deduced HMGS protein were determined by a series of bioinformatics tools.The entire protein-coding cDNA of HMGS was cloned into the expression vector pET-32a(+).Then the recombinant plasmid was transformed into E.coli BL21(DE3)cells.With IPTG induction,SDS-PAGE was used to investigate the situation of expression.Additionally,qRT-PCR technology was performed to measure the transcript levels of HMGS gene in the triterpenoid pathway during different developmental stages of S.baumii.Results The full-length nucleotide sequence of HMGS was 1930 bp,containing a complete open reading frame of 1458 bp which encoded a polypeptide of 485 amino acids.Bioinformatics analysis of the amino acid sequence showed that the molecular weight of encoded protein was 52750,and theoretical isoelectric point was 5.60.This protein was a hydrophilic protein,without transmembrane and signal peptide sequence.The constructed phylogenetic tree showed that HMGS from S.baumii had the highest similarity with HMGS from Fomitiporia mediterranea.The prokaryotic expression vector pET-32a-HMGS was sucessfully obtained.SDS-PAGE results showed that a significant protein band was in consistent with molecular weight of the predicted protein.Moreover,the results showed that the transcript levels of HMGS gene were in dynamic change.The transcript levels in the mycelium stage were higher than that in the fruiting body stage.For instance,the highest transcript level of HMGS was at 14 d and was 2.33-fold higher than the 5 d.Conclusion Molecular characterization of HMGS will be useful for further functional elucidation of the gene involving in triterpenoid biosynthesis pathway in S.baumii.

关 键 词：桑黄 HMGS基因 三萜化合物 表达分析 RACE 

分 类 号：R282.12[医药卫生—中药学]