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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 rLjSABATHMT gene from honeysuckle honeysuckle and compare the expression of homologous gene and intron sequence of SABATH methyltransferase of honeysuckle and honeysuckle honeysuckle, which is the mechanism of active ingredient formation and regulation of honeysuckle Research provides the basis. Methods: According to the gene fragments provided by the cDNA library, specific primers were designed and the cDNA sequence and genomic sequence of rLjSABATHMT gene were cloned from honeysuckle. Bioinformatics analysis of the protein characteristics of the gene, the use of MEGA 5.0 SABATH methyltransferase phylogenetic tree construction, the use of RT-PCR analysis of SABATHMT homology gene in honeysuckle and red and white honeysuckle in different organs, the expression of different florescence , The sequences of intron regions of SABATHMT homologous genes of honeysuckle and honeysuckle were analyzed and compared. Results: The cloned cDNA sequence of rLjSABATHMT gene was 1 251 bp in length with a complete coding sequence encoding a protein of 365 amino acids. The gene protein sequence has a conserved SA-BATHMT domain. Phylogenetic tree shows that it may be salicylic acid / benzoic acid methyltransferase. The results of gene expression analysis showed that the SABATHMT homologues were mainly expressed in flowers. The sequence of intronic region of honeysuckle and honeysuckle honeysuckle was rich in polymorphism, and two SNPs were endemic genotypes of honeysuckle and honeysuckle. The motifs of motif in introns were homologous in two homologous Genes have significant differences. CONCLUSIONS: The variation of SABATHMT intron region may lead to the difference of SABATH methyltransferase expression between Lonicera japonica and Lonicera japonica Thunb.and provide important research bases for the study of the regulation mechanism of Lonicera japonica Thunb.