玉米叶面微生物DNA提取方法及PCR引物的筛选

Screening of DNA Extraction Methods and PCR Primers for Microorganisms from Maize Leaves

传统的微生物培养方法在反映叶面微生态信息上具有局限性,因此逐渐被分子生态学所代替,而获得高质量、大片段、无偏好的总DNA是研究叶面环境微生物的基础。本文采用改进的Sambrook法、CTAB法、改良的化学法以及试剂盒法对玉米生长初期、中期、末期的叶面微生物总DNA进行提取,并对4种方法提取的DNA片段的大小和产量进行综合评价。将得到的DNA用引物357/518以及984/1387对细菌16S rDNA进行扩增;用5种常见引物NS1/fung、ITS1-F/ITS2、NS1/AM1、EF4/fung5、SSU-0817/SSU-1196对真菌18SrDNA进行扩增。结果表明:4种方法提取的DNA片段均适用于PCR,但DNA的得率和纯度有一定差别,其中以试剂盒提取效果最好。PCR结果显示,除化学法外,其他3种方法提取的DNA均能够获得目的条带;并通过比较得出引物984/1387对16S rDNA以及ITS1-F/ITS2对18S rDNA扩增效果较好且扩增量最大。

Traditional isolation and culture method for microorganisms had been found to have limitations in reflecting the whole genome information of phyllospheric microorganisms and have been gradually replaced by molecular ecology methods.The high-quality,large-fragment and unbiased DNA is the important basis for studying phyllospheric microorganisms at molecular level.In this paper,four methods,improved Sambrook method,CTAB method,improved chemical method and Kit method,were adopted respectively to extract the total DNA of microorganisms from maize leaves in early,middle and late growth periods and then the size and output of DNA fragments were evaluated synthetically.The 16S rDNA were amplified by primers 357/518 and 984/1387,meanwhile,the primers NS1/fungi,ITS1-F/ITS2,NS1/AM1,EF4/fung5 and SSU-0817/SSU-1196 were used for the amplification of 18S rDNA.The results showed that all of the four methods could get appropriate fragments for PCR,but the output and purity of DNA were different between different methods and the best was Kit method.Except the improved chemical method,the other methods could get products of PCR.The primers 984/1387 and ITS1-F/ITS2 were the best for amplifying 16S rDNA and 18S rDNA respectively.