芍药ACS基因的克隆及其蛋白质原核表达  

Cloning and Prokaryotic Expression of ACS Gene from Paeonia lactiflora Pall.

作　　者：肖士奎 李芳[1] 张文婷 吕淑芳[1] 史国安[1] 吴疆[3] 范丙友[1] XIAO Shikui;LI Fang;ZHANG Wenting;L Shufang;SHI Guoan;WU Jiang;FAN Bingyou(College of Agriculture,Henan University of Science and Technology,Luoyang 471023,China;SINOMACH TDI International Engineering Co.,Ltd,Luoyang 471003,China;College of Information Engineering,Yulin University,Yulin 719000,China)

机构地区：[1]河南科技大学农学院,河南洛阳471023 [2]中机十院国际工程有限公司,河南洛阳471003 [3]榆林学院信息工程学院,陕西榆林719000

出　　处：《华北农学报》2022年第5期36-44,共9页Acta Agriculturae Boreali-Sinica

基　　金：国家重点研发计划(2018YFD1000400);国家自然科学基金(U1204323);河南省高等学校重点科研项目(20A210010)。

摘　　要：为了研究芍药ACS基因的功能,应用RACE技术从芍药切花品种桃花飞雪中克隆了PlACS基因cDNA序列,利用生物信息学方法对其编码的蛋白质进行了综合分析;以pET32a为骨架,构建了PlACS基因的原核表达载体,建立了PlACS蛋白高效的原核表达体系。结果表明,PlACS cDNA全长1752 bp(GenBank登录号JX512359),共编码492个氨基酸,具有7个保守结构域及活性位点K;分子进化分析表明,PlACS和牡丹ACS亲缘关系最近;二级结构预测发现,PlACS蛋白由α-螺旋、β-折叠、β-转角和无规则卷曲组成,四者比例分别为40.04%,16.26%,6.91%和36.79%;同源建模显示,PlACS蛋白以同源二聚体形式存在。PlACS蛋白最佳诱导表达条件为基因工程菌菌体密度A达0.2时,在菌液中加入终浓度为0.1 mmol/L的IPTG,在37℃诱导2 h。PlACS重组蛋白高效原核表达体系为后续通过变复性获得有生物学活性的PlACS蛋白及其酶学功能鉴定奠定基础。In order to explore the function of ACS gene in herbaceous peony,a full-length cDNA sequence of PlACS cDNA in Paeonia lactiflora was obtained,RACE technique and bioinformatic methods were used to analyze the protein sequence which it encoded.The CDS of PlACS was subcloned,the prokaryotic expression vector of PlACS was constructed based on pET32 a vector,and then the highly efficient prokaryotic expression system was established.The results showed that the total length of PlACS cDNA(GenBank accession JX512359)was 1752 bp,which encoded 492 amino acids.Seven conserved regions and active sites Kwere detected in PlACS protein.Phylogenetic tree analysis showed that PlACS was highest homological with ACS of P.suffruticosa.PlACS protein was determined structurally to be 40.04%α-helix,16.26%β-extended strand,6.91%β-turn and 36.79%random coil.Protein 3 D structure homology modeling predicted that PlACS existed as homodimers.The optimal expression condition of PlACS protein was that when the cell density of genetic engineering strain Areached 0.2,IPTG with a final concentration of 0.1 mmol/L was added,and the recombinant protein was expressed for two hours at 37℃.It was of great significance to acquire PlACS recombinant protein with biological activity by denaturation&renaturation and identify its enzymatic activity in vitro.

关 键 词：芍药 PlACS 基因克隆 序列分析 原核表达 

分 类 号：S682.12[农业科学—观赏园艺] Q78[农业科学—园艺学]