中国中药杂志

2013, v.38(16) 2556-2562

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红白忍冬SABATH甲基转移酶基因克隆及其生物信息学分析
Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis

余晓丹;蒋超;黄璐琦;秦双双;曾祥梅;陈平;袁媛;
YU Xiao-dan1,2,JIANG Chao2,HUANG Lu-qi2,QIN Shuang-shuang2,ZENG Xiang-mei2,CHEN Ping1,YUAN Yuan2*(1.Wuhan Polytechnic University,Wuhan 430023,China; 2.National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

摘要(Abstract):

目的:克隆红白忍冬SABATH甲基转移酶基因(rLjSABATHMT),比较忍冬和红白忍冬SABATH甲基转移酶同源基因表达量和基因内含子序列的差异,为金银花的活性成分形成及调控机制研究提供基础。方法:根据cDNA文库提供的基因片段设计特异性引物,从红白忍冬中克隆获得rLjSABATHMT基因cDNA序列及基因组序列。通过生物信息学对该基因蛋白的特征进行分析,使用MEGA 5.0构建SABATH甲基转移酶系统进化树,利用RT-PCR分析SABATHMT同源基因在忍冬与红白忍冬的不同器官、不同花期的表达量,对忍冬和红白忍冬SABATHMT同源基因内含子区序列进行分析和比较。结果:克隆获得的rLjSABATHMT基因cDNA序列全长为1 251 bp,具有完整编码框,编码365个氨基酸。该基因蛋白序列具有保守的SA-BATHMT结构域,系统进化树结果表明它可能是水杨酸/安息酸甲基转移酶。基因表达分析结果表明SABATHMT同源基因主要在花中表达。忍冬和红白忍冬SABATHMT同源基因内含子区序列具有丰富的多态性,且有2个SNP为红白忍冬的特有基因型;内含子区motif元件中碱基变异在2个同源基因间具有显著差异。结论:SABATHMT同源基因内含子区变异可能导致了忍冬和红白忍冬SABATH甲基转移酶表达差异,为金银花活性成分调控机制研究提供了重要研究基础。
Objective: To clone SABATH methyltransferase(rLjSABATHMT)gene in Lonicera japonica var.chinensis,and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L.japonica with L.japonica var.chinensis.It provide a basis for gene regulate the formation of L.japonica floral scents.Method: The cDNA and genome sequences of LjSABATHMT from L.japonica var.chinensis were cloned according to the gene fragments in cDNA library.The LjSABATHMT protein was characterized by bioinformatics analysis.SABATH family phylogenetic tree were built by MEGA 5.0.The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L.japonica and L.japonica var.chinensis using RT-PCR analysis.Intron sequences of SABATHMT orthologous were also analyzied.Result: The cDNA of LjSABATHMT was 1 251 bp,had a complete coding frame with 365 amino acids.The protein had the conservative SABATHMT domain,and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase.Higher expression of SABATH methyltransferase orthologous was found in flower.The intron sequence of L.japonica and L.japonica var.chinensis had rich polymorphism,and two SNP are unique genotype of L.japonica var.chinensis.The motif elements in two orthologous genes were significant differences.Conclusion: The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L.japonica and L.japonica var.chinensis.These results will provide important base on regulating active compounds of L.japonica.

关键词(KeyWords): 金银花;SABATH甲基转移酶;全长cDNA;内含子
Lonicera japonica;SABATH methyltransferases;full-length cDNA;intSron

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81001605);; 中药制药过程新技术国家重点实验室开放基金项目(SKL2010M0101)

作者(Author): 余晓丹;蒋超;黄璐琦;秦双双;曾祥梅;陈平;袁媛;
YU Xiao-dan1,2,JIANG Chao2,HUANG Lu-qi2,QIN Shuang-shuang2,ZENG Xiang-mei2,CHEN Ping1,YUAN Yuan2*(1.Wuhan Polytechnic University,Wuhan 430023,China; 2.National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

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