基于DNA条形码的桑黄真菌分子鉴定

Molecular identification of ‘Sang Huang’ by DNA barcoding

DNA条形码(DNA barcoding)技术是一种利用生物基因组内短遗传标签进行分类鉴定的方法。桑黄自古以来是一种名贵中药材,而市售桑黄主要依托简单的形态鉴定造成混淆众多。近年来大量研究表明,桑黄类真菌种类至少有7个种,并且其药效也不尽相同。【目的】为更准确、高效对桑黄类真菌进行鉴定,本研究以采集自西藏、吉林和浙江的3种桑黄子实体为材料,筛选最佳DNA条形码与相应PCR条件。【方法】利用组织分离获得3种桑黄纯培养菌株,分别编号为1713、1714和HS菌株。利用ITS、NL、rpb1、rpb2和ef1-α等7组DNA条形码分别对3种桑黄进行PCR扩增、测序、比对和发育树分析,并结合形态学特征,确定其最适DNA条形码和分类学地位。【结果】结果表明,ITS和NL条形码对3种桑黄均能够获得单一目的条带;rpb1Y、rpb2B和rpb2Y条形码特异性低,扩增后获得多个条带;而rpb1B和ef1-α条形码均无法获得条带。通过多序列比对,结合形态分析,确定1713菌株为桑黄纤孔菌(Inonotus sanghuang),而1714和HS菌株均为鲍姆纤孔菌(Inonotus baumii)。【结论】本研究确定ITS和NL为桑黄分子鉴定的最佳DNA条形码,其PCR最适退火温度范围分别为58～59℃和49～50℃,为桑黄真菌快速鉴定提供参考依据。

DNA barcoding is a taxonomic method that uses short genetic markers in an organism's genomic DNA to identify particular species.‘Sang Huang’is a kind of precious traditional Chinese medicinal materials since ancient times.It is easily confused in markets only based on morphological identification.In recent years,a large number of studies have shown that there were at least 7fungal species named‘Sang Huang’with different medicinal activities.【Objective】In the present study,we focused on accurate and efficient identification of‘Sang Huang’mushrooms.Fruiting bodies of three‘Sang Huang’collected from Tibet,Jilin,and Zhejiang were used for screening of the optimum DNA barcodings and their PCR protocols.【Methods】Pure culture 1713,1714,and HS were obtained using the tissue separation method.PCR amplification were performed using seven pairs DNA barcodings of ITS,NL,rpb1,rpb2and ef1-α.The optimal DNA barcodings were determined based on sequence alignment,phylogenetic tree construction,and morphological features.【Results】The results showed that single band product can be amplified using ITS and NL as the primers in the three strains.Multiple nonspecific bands were amplified using rpb1Y,rpb2Band rpb2Yprimers,while no band was obtained using rpb1Band ef1-αprimers.Based on multiple sequence alignment and morphological features,strain 1713was identified as Inonotus sanghuang,strains 1714and HS were both Inonotus baumii.【Conclusion】It suggests that ITS and NL can be used as the optimal DNA barcodings to identify‘Sang Huang’mushrooms,and the optimal range of annealing temperature was 58~59℃,and 49~50℃,respectively.