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比较22份(包括待检材料4份)共计12种药用石斛及密花石豆兰(1份)的叶绿体DNA matK基因序列差异,从分子水平对药用石斛进行鉴定。采用改良的CTAB法提取石斛基因组DNA,PCR扩增,克隆测序法对叶绿体DNA matK基因全序列进行测定,运用BioEdit、MEGA4.0等软件分析序列,构建分子系统树。测得石斛属12种植物的叶绿体matK基因长1410bp,变异位点51个,信息位点11个。除了存在碱基替换的遗传变异外,还存在碱基的插入和缺失。石斛种间以及种内不同居群(品种)间均能在NJ树中明显分化开来,种间平均遗传距离为0.008,种间最大遗传距离为0.014,最小遗传距离为0.003,碱基相差8～20个。种内不同居群(品种)遗传距离为0.001,相差1～5个碱基。并成功地对4种待检种进行了鉴定。利用12种石斛的matK基因全序列数据库及遗传分析软件,通过对待检种的matK基因全序列进行测定,可以在分子水平对石斛不同种及种内不同居群(品种)进行鉴别,为石斛的分子鉴定提供依据。
Comparing the sequence differences of matK gene in chloroplast of 22 medicinal materials including Dendrobium nobile (1) and Medicago sativa (12 samples), including 4 samples to be tested, the medicinal Dendrobium was identified from the molecular level. The modified CTAB method was used to extract the genomic DNA of Dendrobium candidum. The full-length matK gene of chloroplast DNA was amplified by PCR and cloned and sequenced. The molecular phylogenetic tree was constructed by using software such as BioEdit and MEGA4.0. The chloroplast matK gene of 12 species of Dendrobium was 1410 bp in length, 51 of them were mutated and 11 of them were informative. In addition to the genetic variation of base substitutions, there are base insertions and deletions. Dendrobium species and different species within the species were clearly differentiated in the NJ tree, the average genetic distance between species was 0.008, the maximum genetic distance between species was 0.014, the minimum genetic distance of 0.003, the base difference of 8 ~ 20. Within different species (species) within the genetic distance of 0.001, a difference of 1 to 5 bases. And successfully identified four species to be seized. Based on the matK gene complete sequence database and genetic analysis software of 12 kinds of Dendrobium, the whole matK gene sequence of the tested species can be used to identify different species and species of Dendrobium, Molecular identification provide the basis.