杂草稻nrDNA ITS片段的PCR扩增条件优化及测定

Optimizing of the PCR Amplification Conditions and Sequencing the Weedy Rice nrDNA ITS

对杂草稻的nrDNA ITS片段的PCR扩增条件进行了优化并测定,建立的PCR最优体系为:20μL反应体系含1μL模板DNA,0.125 mmol/L dNTPs,0.5μmol/L正反向引物,1 UTaq酶,2.0μL 10×TaqPCR Buffer;退火温度为57℃。这样的条件下充分保证了ITS PCR产物的质量和纯度要求,直接测序结果为600 bp左右,与网上结果十分类似,表明结果准确可靠。这些ITS片段的系统学信息将为杂草稻的起源进化提供有力的分子水平证据。

The PCR conditions of the weedy rice nrDNA ITS were optimized in this study, as a result, the PCR reactions were camed out as follows： each reaction mixture contained 1 μL genomic DNA,0. 125 mmol/L each dNTPs,0.5 mmol/L forward and reverse primer,2.0 μL 10 × PCR reaction Buffer,and the final reaction was adjusted to 20 μL with sterilized ddH2O. The annealing temperature was 57 ℃. It can assure that PCR products quality and purity using these optimal conditions. The length of the ITS fragments was about 600 bp using the direct sequencing methods and consistent with the genebank data. The phylogenetic information of the weedy rice ITS would conveniently provide the direct molecular evidence to clarify its origin and evolution.