PCR产物直接测序还是克隆测序?——密叶杉属rDNA ITS序列的测定方法

通过PCR产物直接测序和克隆测序对三种密叶杉属 (Athrotaxis)植物rDNA内转录间隔区 (ITS)及5 .8SrDNA序列进行了测定与分析。实验表明A .selaginoidesrDNA重复序列间的纯合程度很高 ,对PCR产物直接测序就可以测定其ITS区序列。而A .laxifolia、A .cupressoides的ITS1重复序列间的纯合程度较低 ,各重复单位间序列存在插入 /缺失 ,只有对PCR产物进行克隆测序才能确定其序列。A .laxi folia、A .cupressoides的ITS2区尽管也存在多态性 ,但不同重复序列的浓度比较平均 ,对PCR产物直接测序就可确定重复序列间的变异情况。本实验表明尽管是同一属的三种植物 ,但其rDNA重复序列间的纯合程度不同 ,同一植物ITS的不同区域 ,其重复序列间的纯合程度也不同 ,针对不同的ITS片段可采用不同的方法以测定其序列。

Direct Sequencing of PCR Products or Sequencing by Cloning PCR Products-Method of Determining Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA in Athrotaxis

The results of direct sequencing of PCR products or sequencing by cloning PCR products of the internal transcribed spacers and the 5.8S coding region of nuclear ribosomal DNA in Athrotaxis were compared and analyzed. The rDNA repeat units of A. selaginoides have been homogenized, so its ITS region sequences were determined by direct sequencing of PCR products, but the rDNA repeat units of A. laxifolia and A. cupressoides have not been homogenized, there are many polymorphic sites and insersion/deletions in the ITS1 regions, so their ITS1 sequences were determined by cloning PCR products and then sequencing. There are polymorphic sites but no insersion/deletions in ITS2 region of A. laxifolia and A. cupressoides , so the direct sequencing of PCR products can also be used to display variable nucletides at one site. The results in our studies show that the different ITS regions of Athrotaxis have been homogenized at different extent, so the methods of direct sequencing of PCR products or sequencing by cloning PCR products can be selected to determine their sequences.