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本文基于兰科已有的LEAFY(LFY)同源基因保守区设计引物,对细茎石斛(Dendrobium moniliforme(L.)Sw.)的该基因进行克隆及序列分析。通过普通PCR及Tail-PCR技术得到LFY同源基因——DenLFY,全长共3 575 bp,包含3个外显子和2个内含子。将DenLFY基因外显子序列提交到NCBI进行BLAST比对,发现该基因与兰科其他植物同源基因序列相似度很高,均达80%以上,其中与蝴蝶兰(Phalaenopsis hybrid cultivar)LFY同源基因(PhalLFY)和文心兰(Oncidium Gower Ramsey)LFY同源基因相似度分别达84%和90%。应用MP的方法,根据不同物种LFY同源基因进行系统发生关系分析,发现细茎石斛与文心兰、蝴蝶兰亲缘关系最近。氨基酸序列比对分析结果表明,该基因C端编码的氨基酸相对更为保守。从DenLFY基因外显子和内含子单独分析来看,外显子的获得及其编码氨基酸的推测对于LFY基因功能的研究是一个开端,而内含子2在细茎石斛和铁皮石斛(Dendrobium officinale Kimura et Migo)中存在长度差异,在石斛药材鉴定方面有着很大的应用潜力。
In this paper, we cloned and sequenced the gene of Dendrobium moniliforme (L.) Sw. Based on the conserved region of LEAFY (LFY) homologous genes in Orchidaceae. The LFY homologous gene, DenLFY, was obtained by PCR and Tail-PCR. The full-length cDNA was 3575 bp in length and contained 3 exons and 2 introns. The exon sequence of DenLFY gene was submitted to NCBI for BLAST analysis. The results showed that there was high homology between homologous genes and other homologous genes of Orchidaceae, which reached more than 80%, which was homologous to LFY of Phalaenopsis hybrid cultivar The homologies of the LFY homologues of the gene (PhalLFY) and Oncidium Gower Ramsey were 84% and 90%, respectively. According to phylogenetic analysis of LFY homologous genes of different species, we found that Phyllanthus nobile had the closest genetic relationship with Oncidium and Phalaenopsis. Amino acid sequence alignment analysis showed that the gene C-terminal amino acid encoding is relatively more conservative. From the separate analysis of exon and intron of DenLFY gene, the study of exon gain and deduced amino acid coding for LFY gene function is the beginning, and intron 2 is the most important gene in Dendrobium officinale and Dendrobium officinale Kimura et Migo) in the length of differences in the identification of Dendrobium medicinal herbs has great potential.