摘要
目的:克隆分析夏枯草中对羟基苯丙酮酸双氧化酶基因PvHPPD,并对其编码蛋白进行原核表达与纯化,为日后探索对羟基苯丙酮酸双氧化酶的生物学作用提供依据。方法:基于夏枯草转录组数据库,利用同源比对和电子克隆得到PvHPPD cDNA序列,设计引物扩增PvHPPD cDNA,并进行生物信息学分析。将PvHPPD cDNA序列连接到pMAL-c5x载体上融合麦芽糖标签,转化BL21(DE3)表达菌中,IPTG诱导蛋白表达并进行纯化。结果:PvHPPD的cDNA序列长1850 bp,ORF序列长1452 bp,编码483个氨基酸,编码蛋白为亲水性非跨膜蛋白。多序列比对分析表明,该基因具有HPPD蛋白特有的保守结构域,保守区域多集中在C端,N端多变化。系统进化分析表明,PvHPPD基因表达蛋白与唇形科植物聚为一组,并与虎尾紫苏的同源性达85%。体外蛋白表达发现该蛋白在上清液和沉淀(包涵体)中均有表达,沉淀中的表达更多。结论:该研究成功获得PvHPPD基因,并掌握了该基因编码蛋白的生物信息学特征及体外表达体系,该结果将有利于后续深入研究PvHPPD基因及其在迷迭香酸生物合成途径中的功能和调控机制。
Objective:To clone and analyze para-hydroxyphenylpyruvate bioxidase gene PvHPPD from Prunella vulgaris,and to express and purify its encoded protein,so as to provide the basis for exploring the biological function of para-hydroxyphenylpyruvate bioxidase in future.Methods:PvHPPD cDNA sequence was obtained by homology alignment and electronic cloning based on the transcripome database of Prunella vulgaris.Primers were designed to amplify PvHPPD cDNA,and bioinformatics analysis was performed.PvHPPD cDNA sequence was attached to the pMAL-c5x vector and fused maltose tag was transformed into BL21(DE3)expressing bacteria,and IPTG-induced protein expression was performed and purified.Results:The cDNA sequence length of PvHPPD was 1850 bp,ORF sequence length of PvHPPD was 1452 bp,encoding 483 amino acids,and the encoding protein was a hydrophilic non-transmembrane protein.Multiple sequence alignment analysis showed that the gene had the unique conserved domain of HPPD protein,and the conserved domain was mainly concentrated in the C-terminal and varied in the N-terminal.Phylogenetic analysis showed that the PvHPPD gene expression protein was clustered into a group with the Labiaceae plants,and the homology with Perilla frutescens var.hirtella was 85%.In vitro protein expression,found that the protein was expressed in both supernatant and precipitate(inclusions),with more expression in precipitate.Conclusion:In this study,PvHPPD gene is successfully cloned,and the bioinformatics characteristics and in vitro expression system of the protein encoded by the gene are mastered.The results are benifit to promote further research on the function and regulatory mechanism of PvHPPD gene and it pathway in rosmarinic acid biosynthesis.
作者
蓝婷婷
赵兵菲
吴传梅
梁潘
杨楚楚
谭小姚
汝梅
LAN Ting-ting;ZHAO Bing-fei;WU Chuan-mei;LIANG Pan;YANG Chu-chu;TAN Xiao-yao;RU Mei(Guangxi University of Chinese Medicine,Nanning 530200,China)
出处
《中药材》
CAS
北大核心
2024年第3期581-586,共6页
Journal of Chinese Medicinal Materials
基金
广西科技基地和人才专项(桂科AD19245087)
2021年大学生创新创业训练计划项目(202110600036)
2023年大学生创新创业训练计划项目(S202310600141)。
作者简介
蓝婷婷(2000-),女,在读本科生,专业方向:中药资源,E-mail:258305196@qq.com;通讯作者:汝梅,Tel:0771-4928552,E-mail:rumei2015@163.com。
