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5.8 S rDNA序列进行了测定与分析。实验表明A. selaginoides rDNA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. laxifolia、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.