应用克隆文库研究玉米根系AMF多样性方法的建立

以玉米根系为例,建立植物根系丛枝菌根18SrRNA基因克隆文库构建的方法。CTAB法提取的丛枝菌根真菌总DNA能够满足后续PCR扩增的要求。运用真菌引物GeoA2/Geo11及AMF特异引物AM1、AM2、AM3/NS31进行巢式PCR扩增,获得的产物连接转化入大肠杆菌构建18SrRNA部分基因克隆文库。通过限制性内切酶Hinf I、TaqI对随机挑选的50个克隆子进行ARDRA(Amplified Ribosomal DNA Restriction Analysis)分析,共获得11个差异图谱。每个图谱选择1个代表测序,结果表明,3个序列为玉米自身未知基因,其余8个都为Clomus属序列。文库评价统计结果发现,AMF克隆文库的Coverage值为72.4%,Rarefaction曲线趋于平缓,所建立文库的容量较为充足,能较全面地代表玉米根系AMF的多样性。

Species Diversity of AMF Community Colonized in Maize Roots by Clone Library

To study species diversity of AMF community colonized in maize roots, AMF 18S rRNA gene clone library construction methods have been established. Genomic DNA of AMF from conservatory cultured maize roots have been isolated by CTAB DNA extraction method, nested PCR was carried out for AMF 18S rDNA amplification using different primers (GeoA2/Geo11, AM1, AM2, AM3/NS31), after library construction and screening by ARDRA (Amplified Ribosomal DNA Restriction Analysis) using Hinf I, TaqI digestion, eleven represent clones have been sequenced. Three of the sequences were function unknown maize genes and the rest belong to Clomus genus. Several softwares were used to calculate diversity index and estimate the library capability. The coverage value was 72.4% and the rarefaction curve showed a tendency to saturation revealed the capacity of this library was enough to represent the AMF diversity. This preliminary attempt provided a valid method for diversity of AMF study and would be very meaningful for recognizing and utilizing these important microbes.