几种灵芝rDNA基因间隔序列分析与鉴定

Analysis and identification of rDNA ITS sequences of several Ganoderma species

摘要:

目的  基于基因间隔序列（ITS）序列对灵芝进行分类。方法  培养灵芝、提取DNA、优化聚合酶链反应（PCR）体系、纯化与克隆、测序、构建遗传进化树、进行遗传分析。结果  经过一系列的梯度实验得知优化的PCR体系为25μl反应混合物中含模板DNA 25 ng、Mg2+为2.0 mmol/L、dNTPs 200μmol/L、Taq 1.5 U、引物为20 pmol；最佳反应程序为93℃预变性4 min，93℃变性40 s，59℃退火40 s，72℃延伸1 min，共35个循环，72℃继续延伸7 min，4℃无限循环。所采用的HZ002、DZ003、NZ004和XC005在遗传进化树上面十分接近，说明其可以划分为同一菌属，同属于灵芝菌属。结论  基于ITS序列对灵芝进行分类，与其他的分子标记技术所取得的分类结果一致，证实依据ITS序列来对真菌菌属进行分类是十分合理的。

Abstract:

Objective To classify Ganoderma based on internal transcribed spacer (ITS) sequences. Methods Several species of Ganoderma were cultivated. DNA was extracted. PCR system was optimized followed by purification and cloning, and sequencing. Then phylogenetic tree was constructed, and genetic analysis was made. Results The whole PCR system was optimized which included 25 μl reaction mixture containing template DNA 25 ng, Mg2+ 2.0 mmol/L, dNTPs 200 μmol/L, Taq 1.5 U, primer 20 pmol. The optimum reaction program was pre-denaturation at 93℃ for 4 min, denaturation at 93℃ for 40 s, annealing at 59℃ for 40 s, prolongation at 72℃ for 1 min for 35 cycles; then final prolongation at 72℃ for 7 min followed by 4℃ infinite loop. Adopted HZ002, DZ003, NZ004 and XC005 were very close in the phylogenetic tree, indicating that they belonged to the same genus, Ganoderma. Conclusions ITS sequence classification is consistent with the traditional taxonomy and classification obtained with other molecular markers, which is very reasonable for classification of fungal genus.