摘要
目的克隆地涌金莲Musella lasiocarpa中O-甲基转移酶(O-methyltransferase,OMT)基因,并对其进行生物信息学及表达模式分析。方法基于地涌金莲转录组数据,筛选并原核表达了3条MlOMTs酶基因(MlOMT1、MlOMT2和Ml OMT3)。利用软件对其进行生物信息学分析,利用MEGA11构建系统发育树,采用实时荧光定量PCR检测基因表达模式。结果PCR扩增得到3条MlOMTs基因;3个MlOMT蛋白长度为289~400个氨基酸,相对分子质量在3147360~4526756,3个蛋白均为不稳定的亲水性蛋白,其中MlOMT1和MlOMT2不含跨膜区,MlOMT3存在2个跨膜螺旋,其N端位于胞质内。3个蛋白主要定位于叶绿体,不含有信号肽,主要由α-螺旋和无规则卷曲构成,含有SAM依赖的甲基转移酶超家族的保守结构域,属于SAM依赖的甲基转移酶超级家族。系统进化分析表明,MlOMT1与来源于大麦Hordeum vulgare的Hv7OMT和粳稻Oryzasativasubsp.japonica的OsOMT17聚为一支,MlOMT2与冰叶日中花Mesembryanthemum crystallinum的McPFOMT聚为一支,而MlOMT3与拟南芥Arabidopsis thaliana的AtCCoAOMT1聚为一支,表明它们亲缘关系较近,推测它们具有类似的生物学功能。通过对表达条件的初步优化,重组工程菌pMal-c4X-MlOMT1和pMal-c4XMlOMT2在大肠杆菌BL21(DE3)中实现蛋白可溶性表达。组织特异性表达分析结果显示,MlOMT1在地涌金莲不同组织相对表达量为:叶片>苞片>种子,MlOMT2和MlOMT3相对表达量为:苞片>种子>叶片。结论成功克隆获得地涌金莲3条MlOMTs基因并进行生物信息学分析和原核表达,为后续酶的功能表征奠定基础。
Objective To clone the O-methyltransferase(OMT)genes in Musella lasiocarpa and analyze their bioinformatics information and expression patterns.Methods Three MlOMTs enzyme genes(MlOMT1,MlOMT2 and MlOMT3)were screened based on the transcriptomic data of M.lasiocarpa and prokaryotically expressed.Online software were used for bioinformatics analysis.Phylogenetic tree was constructed using MEGA11.Real-time fluorescence quantitative PCR was conducted to detect gene expression patterns.Results Three MlOMTs were cloned by PCR.The lengths of the three MlOMT proteins ranged from 289 to 400 amino acids,and the range of the predicted molecular weight was from 3147360 to 4526756.Three MlOMTs were unstable hydrophilic proteins.Both MlOMT1 and MlOMT2 lacked transmembrane regions,whereas MlOMT3 had two transmembrane helices with its N-terminal located in the cytoplasm.All the three proteins were mainly localized in chloroplasts,without signaling peptides.They mainly consisted ofα-helical and irregular coiled structures,and contained conserved domains of the SAM-dependent methyltransferase superfamily.Phylogenetic analysis revealed that MlOMT1 clustered with Hv7OMT from Hordeum vulgare and OsOMT17 from Oryza sativa subsp.Japonica,while MlOMT2 clustered with McPFOMT from Mesembryanthemum crystallinum.Additionally,MlOMT3 clustered together with AtCCoAOMT1 from Arabidopsis thaliana.The close relationships mentioned above suggested they might have similar biological functions.The soluble protein expression of recombinant vectors pMal-c4X-MlOMT1 and pMal-c4X-MlOMT2 in BL21(DE3)was achieved through preliminary optimization of the expression conditions.Tissue-specific expression analysis showed that relative expression levels of MlOMT1 varied across different tissues as follows:leaves>bracts>seeds;whereas relative expression levels of MlOMT2 and MlOMT3 were found as bracts>seeds>leaves.Conclusion The cloning,bioinformatics analysis and expression analysis of three MlOMTs of M.lasiocarpa was completed,which lays the foundation for characterizing their functions.
作者
吴君芝
林谷音
王帆
李丕睿
吕威
赵万里
陈雨
WU Junzhi;LIN Guyin;WANG Fan;LI Pirui;LYU Wei;ZHAO Wanli;CHEN Yu(Nanjing University of Chinese Medicine,Nanjing 210023,China;Jiangsu Province and Chinese Academy of Sciences(Nanjing Botanical Garden Mem.Sun Yat-Sen),Nanjing 210014,China)
出处
《中草药》
CAS
CSCD
北大核心
2024年第15期5212-5221,共10页
Chinese Traditional and Herbal Drugs
基金
国家自然科学基金面上项目(32070360)。
关键词
地涌金莲
甲基转移酶
基因克隆
实时荧光定量PCR
生物信息学分析
Musella lasiocarpa(Franch.)C.Y.Wu ex H.W.Li
methyltransferase
gene cloning
quantitative real-time PCR
bioinformatics analysis
