北沙参异戊烯基转移酶GlIPT1基因的克隆及生物信息学分析  

Cloning and Bioinformatics Analysis of Isopentenyltransferase Gene in Glehniae Radix(GlIPT1)

作　　者：陈泓宇 任宏伟 张玉喜[1] 王卫青 谭玲玲[1] 高婷[1,2] Chen Hongyu;Ren Hongwei;Zhang Yuxi;Wang Weiqing;Tan Lingling;Gao Ting(Key Laboratory of Plant Biotechnology in Universities of Shandong Province,College of Life Sciences,Qingdao Agricultural University,Qingdao 266109,China;Shanghai Key Laboratory of Plant Functional Genomics and Resources,Shanghai Chenshan Botanical Garden,Shanghai 116021,China)

机构地区：[1]青岛农业大学生命科学学院山东省高校植物生物技术重点实验室,青岛266109 [2]上海市资源植物功能基因组学重点实验室,上海116021

出　　处：《世界科学技术-中医药现代化》2020年第4期1176-1184,共9页Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology

基　　金：国家自然科学基金委员会青年基金项目(81903748):黄芩黄酮合成相关R2R3-MYB转录因子可变剪切异构体的功能及机理研究,,负责人:高婷;上海市资源植物功能基因组学重点实验室开放课题(PFGR201703):珊瑚菜呋喃香豆素次生代谢途径关键酶异戊烯基转移酶(IPT)基因的鉴定研究,基金立项单位:上海市资源植物功能基因组学重点实验室(上海辰山植物园),主持人:高婷

摘　　要：目的挖掘参与北沙参(Glehniae Radix)香豆素合成的关键酶异戊烯基转移酶(Isoprenyltransferase,IPT)基因,进一步分析该基因的序列特征及表达量模式。方法本研究摸索了一种北沙参基原植物珊瑚菜(Glehnia littoralis Fr.Schmidt ex Miq.)无菌苗制备方法,并且在珊瑚菜转录组测序数据的基础上,使用茉莉酸甲酯(MeJA)处理筛选出表达量上调的候选基因序列;利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)技术对GlIPT1基因cDNA序列进行全长克隆;根据所得的氨基酸序列进行蛋白质理化性质、二级结构、三级结构分析;利用Clustalx及MEGA 6.0构建NJ系统进化树;利用实时荧光定量PCR(RT-qPCR)方法检测该基因的组织特异性表达。结果GlIPT1基因cDNA序列全长为1296 bp,编码306个氨基酸残基,蛋白相对分子质量为34409.30 Da,等电点为5.93,属于P-loop_NTPase超家族,为亲水性蛋白。系统进化分析表明,GlIPT1与伞形科植物胡萝卜Daucus carota subsp.sativus IPT蛋白亲缘关系较近。荧光定量PCR结果显示:GlIPT1基因在珊瑚菜的花中表达量最高,其次是根,在叶中表达量较低。结论本实验为进一步研究北沙参的分子育种和利用生物技术提高香豆素产量奠定了基础,具有重要的理论价值和良好的应用前景。Objective To screen the key enzyme isoprenyltransferase(IPT)gene involved in coumarin synthesis in Glehniae Radix and further analysis of the sequence characteristics and expression pattern of the gene.Methods Based on the previous transcriptome sequencing date of Glehnia littoralis and analysis of expression after treatment with methyl jasmonate(MeJA),one IPT candidate sequence was screened.Then the 3′cDNA end of GlIPT1 was cloned by rapid amplification of cDNA ends(RACE)and the full-length cDNA sequence was assembled by DNAMAN software,and the encoded protein was analyzed by the bioinformatics tools.The issue specific expression of GlIPT1 was also detected using qPCR.Results It showed that the full-length cDNA sequence of GlIPT1 gene was 1296 bp,which encoding 306 amino acid residues.The relative molecular mass of the protein was 34409.30 Da and the isoelectric point was 5.93.We found that GlIPT1 belongs to the P-loop_NTPase superfamily and it is a hydrophilic protein.Phylogenetic analysis indicated that GlIPT1 is genetically closely related to the IPT of Dauricus carota subsp.sativus.The results of RT-qPCR showed that the highest expression of GlIPT1 was found in flowers,followed by roots,and the lowest was in leaves.Conclusions This experiment provides a basis for further research on molecular breeding of Radix Glehniae and using biotechnology to increase coumarin production.It has important theoretical value and good application prospects.

关 键 词：北沙参 异戊烯基转移酶基因 RACE 生物信息学分析 组织特异性表达 

分 类 号：R281[医药卫生—中药学]