cpDNA psbA-trnH在硬叶兜兰分子进化中的应用价值

cpDNA psbA-trnH基因被认为是被子植物叶绿体基因组中变异位点最多的序列和进化速率最快的叶绿体间隔子之一,该序列可运用于药材种或品种、产地鉴定、系统进化分析。为了更好地了解兜兰属植物的特征和系统进化研究价值,本研究通过建立硬叶兜兰PCR扩增反应体系,对其叶绿体转录间隔基因(psbA-trnH)扩增效果和测序变异进行研究。结果表明,采用60 ng/10μL的DNA模板、2.5 mmol/L的MgCl₂、各0.5μmol/L的引物浓度、0.6 U/10μL的DNA聚合酶和0.3 mmol/L dNTPs,退火温度为58.8℃的反应体系有较好的扩增效率,平均测序成功率为53.03%。该基因长度为684~955 bp,平均长度为835 bp,长度变异系数为9.32%,共检出49个核苷酸变异位点,该基因在种水平的变异较小,在亚科间有不同程度的变异。硬叶兜兰的psbA-trnH序列在居群水平上表现出一定的变异,但平均测序成功率不高,仅为53.03%,在一定程度上限制其应用。

Application Prospect of cpDNA psbA-trnH in Molecular Evolution of Paphiopedilum micranthum

cpDNA psbA-trnH is considered as one of the fastest evolution sequences with much more mutation sites in the chloroplast genome of angiosperms.Therefore,it can be used in the medicine identification of indistinguishable species or variety,origin identification and phylogenetic analysis.In order to analyze the amplification efficiency of cpDNA psbA-trnH and its sequence variation of Paphiopedilum micranthum based on an optimized PCR reaction system.The results showed that the optimal system included 60 ng/10μL DNA template,2.5 mmol/L MgCl₂,0.5μmol/L primers concentration,0.6 U/10μL DNA polymerase,0.3 mmol/L dNTPs with the annealing temperature at 58.8℃.PCR system could effectively amplify cpDNA psbA-trnH gene from Paphiopedilum micranthum which had been sequenced successfully with an average percentage of 53.03%.Its length ranged from 684~955 bp with an average of 835 bp.Variation coefficient of sequence length attained to 9.32%.Its nucleotide sequence had 49 variation loci.There was no much difference at species level but a little different at subfamily level.It also indicated that the application of psbA-trnH for populations'genetic structure was limited because of a low percentage of sequencing success of 53.03%,although there were more mutant loci at population level.