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目的对石斛属叶绿体IRa(trn V-GAC~trn R-ACG)序列进行系统发育和变异位点分析,深入了解石斛属10个物种此序列所含9个基因的异同,为石斛属植物的系统进化历程及DNA条形码的研究作重要探索。方法用改良试剂盒法提取石斛属10个样本总DNA,自行设计引物,采用聚合酶链式反应(polymerase chain reaction,PCR)及T载体对PCR产物进行克隆转化,扩增石斛属叶绿体IRa序列的DNA序列,并与NCBI数据库进行序列比对完成序列拼接;绘制此区域序列的基因结构图,对10条目的序列进行差异位点、遗传距离、系统进化关系等分析。结果目的序列长约7 800 bp,10条目的序列共存在55个差异位点,在基因trn V-GAC、rrn16、trn I-GAU、trn A-UGC、orf42、rrn23及基因间隔区上分布的个数分别是1、8、12、6、1、9和18个;系统发育分析将石斛属10个样本聚为4个类群。结论建立了测定石斛属叶绿体IRa序列的方法,对石斛属这一序列的系统发育和变异位点分析表明,此区域序列丰富,且较为集中分布的变异位点将为石斛属DNA条形码的进一步研究、石斛属资源的道地性评价及相关的种质资源鉴定提供较好的理论依据。
Aim To investigate the phylogeny and variation of IRa (trn V-GAC trn R-ACG) sequences in Dendrobium, and to understand the similarities and differences of 9 genes contained in 10 sequences of Dendrobium, Evolutionary history and DNA bar code as an important exploration. Methods Total DNA was extracted from 10 samples of Dendrobium by modified kit method. Primers were designed by PCR. The PCR products were cloned and transformed by polymerase chain reaction (PCR) and T vector. DNA sequence, and the NCBI database sequence alignment to complete the sequence splicing; draw the gene sequence map of the region, the sequence of 10 different sites, genetic distance, phylogenetic relationships and other analysis. Results The sequence of the target was about 7 800 bp. There were 55 different sites in 10 sequences, which were distributed in the genes trn V-GAC, rrn16, trn I-GAU, trn A-UGC, orf42, rrn23 and intergenic spacer The numbers were 1, 8, 12, 6, 1, 9 and 18, respectively. Phylogenetic analysis clustered 10 samples of Dendrobium into 4 groups. Conclusion The method of determining the IRa sequence of the chloroplast of Dendrobium was established. The phylogenetic analysis and the variation of the sequence of Dendrobium showed that this region was rich in sequence and the more concentrated sites would be the further study of Dendrobium DNA barcode , Dendrobium resources of the authenticity evaluation and related germplasm resources identification to provide a better theoretical basis.