摘要
目的:为了研究当归阿魏酸生物合成途径的功能基因,从当归中克隆了一条当归咖啡酸-O-甲基转移酶基因(AsCOMT),进行生物信息学分析、基因表达模式分析及原核表达。方法:首先依据当归COMT基因的序列(GenBank注册号KP188587),利用PCR的方法克隆该基因的cDNA全长。对AsCOMT基因进行实时荧光定量PCR分析。采用无缝克隆技术构建AsCOMT基因的原核表达载体,通过热击法转化大肠杆菌BL21(DE3)。进一步利用异丙基-β-D-硫代半乳糖(IPTG)诱导大肠杆菌表达重组蛋白,并筛选出最佳IPTG诱导表达条件。结果:AsCOMT基因的开放阅读框为1098 bp,编码365个氨基酸,蛋白分子量约为40.230 kDa,等电点为5.43。AsCOMT蛋白含有1个S-腺苷-L-甲硫氨酸结合位点,属于COMT蛋白家族成员。通过实时荧光定量表达分析表明,AsCOMT基因在当归的不同组织均有表达,根中表达量最高,其次是叶片,叶柄中表达量最低。原核表达载体pGEX-4T-3-AsCOMT在E.coli BL21(DE3)中大量表达当归AsCOMT蛋白,且经SDS-PAGE分析显示其相对分子质量约为40.23 kDa,其最优表达条件为25℃、1.0 mmol/L IPTG终浓度、诱导8 h,得到的当归AsCOMT蛋白主要以可溶性形式存在。结论:本研究成功克隆了AsCOMT基因,且该基因的表达具有组织特异性。构建的AsCOMT原核表达菌株在最适IPTG诱导表达条件下能大量表达当归COMT蛋白,为研究COMT基因在当归阿魏酸生物合成途径中的功能奠定了基础。
Objective:In order to investigate the functional genes involved in ferulic acid biosynthesis pathway of Angelica sinensis(Oliv.)Diels,a caffeic acid O-methyltransferase gene(AsCOMT)was isolated from A.sinensis.Meanwhile,the bioinformatic analysis,expression patterns and prokaryotic expression of AsCOMT gene were carried out.Methods:Based on the sequence of COMT gene of A.sinensis(GenBank Registration No.KP188587),the full length of cDNA of AsCOMT gene was cloned by PCR.The expression pattern of AsCOMT gene in different tissues was detected by real-time fluorescence quantitative PCR.The recombinant expression plasmid of AsCOMT gene was constructed by In-fusion cloning technology and it was transformed into E.coli BL21(DE3)by heat shock method.Furthermore,the recombinant AsCOMT protein was expressed in E.coli BL21(DE3)cells under IPTG induction and the optimal expression conditions were screened.Results:The ORF of AsCOMT gene was 1098 bp,which encodes 365 amino acids.The molecular weight of protein encoded by AsCOMT gene was about 40.230 kDa and its isoelectric point is 5.43.AsCOMT protein contained a SAM binding site,which belonged to the COMT protein family.The results of real-time PCR indicated that AsCOMT gene was expressed in high transcript level in roots,lower level in leaves and the lowest level in petioles of A.sinensis.In E.coli BL21(DE3)the recombinant expression plasmid(pGEX-4T-3-AsCOMT)expressed a large amount of AsCOMT protein and its results of SDS-PAGE analysis showed the relative molecular weight of AsCOMT protein was about 40.230 kDa,the optimal expression conditions of AsCOMT protein was 1.0 mmol/L IPTG at 25℃for 8 h,which was mainly in the form of soluble protein.Conclusion:The AsCOMT gene was cloned successfully and its expression had specificity in different tissues.Moreover the pGEX-4T-3-AsCOMT in E.coli BL21(DE3)could express a large number of AsCOMT protein under the optimal conditions.The results of this study provide a foundation for functional characterization of AsCOMT gene involved in ferulic acid biosynthesis pathway of A.sinensis.
作者
张培
侯云龙
苏敏
孙利
徐登封
宋涛
周璐恒
赵韶华
Zhang Pei;Hou Yunlong;Su Min;Xu Dengfeng;Song Tao;Zhou Luheng;Zhao Shaohua(Institute of Chinese Materia Medica of Chengde Medical University, Chengde 067000, China;Shijiazhuang Second Hospital, Shijiazhuang 050051, China;Shijiazhuang Yiling Pharmaceutical Co., Ltd., Shijiazhuang 050051, China)
出处
《中国野生植物资源》
CSCD
2021年第1期20-28,共9页
Chinese Wild Plant Resources
基金
国家重大新药创新(2019ZX09201005-002-001)。
关键词
当归
COMT
荧光定量PCR
原核表达
最佳表达条件
Angelica sinensis
Caffeic acid O-methyltransferase
Real-time PCR
Prokaryotic expression
Optimal expression condition
