中药材川贝母标准品特异DNA指纹片段的克隆及鉴定

Cloning and Identification of a Specific Standard DNA Fingerprint Fragment from Chinese Herbal Medicine Fritillaria cirrhosa D.Don

目的建立一种能够有效保存川贝母标准品DNA指纹片段的方法。方法采用自主研制的川贝母DNA检测试剂盒提取基因组DNA,体外进行川贝母特异DNA片段克隆,与载体结合并导入生物宿主细胞,通过宿主细胞自我复制产生大量川贝母特异DNA克隆,继而采用质粒提取试剂盒将质粒提取出来,应用PCR及酶切方法检查并鉴定插入片段是否正确。将携带正品川贝母特异DNA序列的菌落置-80℃冰柜保存,分别以1个月、2个月、3个月和6个月为时间点进行复苏,进行稳定性考察。结果转入细胞内的川贝母DNA克隆经PCR扩增后,所得到的琼脂糖凝胶电泳图谱条带清晰,并且进行多次实验结果均一致。4个时间点复苏后的菌落进行PCR扩增后,其琼脂糖凝胶电泳检测图谱中,条带明亮且清晰。结论采用分子克隆技术保存川贝母特异基因片段的方法是可行且有效的,并且稳定性良好,可作为建立中药DNA指纹鉴定数据库的有效依据,方法简单明了,且结果可靠。

OBJECTIVE To establish a standard method for preserving the Fritillaria cirrhosa D. Don DNA fingerprint fragments.METHODS Independently developed F. cirrhosa test kit was used to extract genomic DNA. Specific F. cirrhosa DNA fragments were cloned in vitro,and ligated into a carrier vector then transformed into a self replicating host cells to produce a large amount of specific F. Cirrhosa DNA. Plasmid DNA was extracted using Plasmid Extraction Kit and confirmed by PCR or restriction digestion of the insert. Bacterial colonies carrying authentic Sichuan F. cirrhosa specific DNA sequences were stored at- 80 ℃. Stability was monitored at intervals of 1,2,3 and 6 months after DNA recovery using the boiling method of genomic DNA extraction. RESULTS The amplified F. cirrhosa DNA clones showed clear bands in the electrophoresis,and had stable results in the repeated verification test.The amplified clones from resuscitated bacterial colonies which had been stored for 1,2,3,6 months at- 80 ℃ still displayed bright and clear bands after electrophoresis. CONCLUSION It is feasible and effective to preserve DNA fingerprint of F. Cirrhosa by the established method,and this simple and reliable method can be used as the basis of establishing the new genes database of traditional Chinese medicine.