Establishment, Optimization and Application of cDNA-SCoT Reaction System in Sugarcane
Gene differential expression analysis by cDNA-SCoT is a newly developed technique in plants.In this study,the root tip of sugarcane variety GT11 was used as the experiment material.We isolated the total RNA of sugarcane root tip and it was reversely transcripted into the first strand cDNA.And then,we optimized SCoT-PCR amplification system of sugarcane in five factors such as template cDNA,SCoT primers,Taq DNA polymerase,dNTPs and Mg2 + to establish an optimal reaction system of sugarcane,which was used to isolate differentially expressed genes in sugarcane root tip induced by 25% PEG6000.The results showed that all of the five factors affected the PCR amplification and the optimal 20 μL PCR reaction mixtures in sugarcane was template cDNA 80 ng,SCoT primers 1.0 μmol/L,Taq DNA polymerase 2.0 U,dNTPs 200 μmol/L and Mg2+1.8 mmol/L.The amplification results of SCoT primers indicated higher genetic diversity at the polymorphic rate of 46.9%,and the average bands of each primer were 30 bands.What should be stressed is that we obtained a total of 45 differentially expressed bands in sugarcane root tip induced by 25% PEG.The cDNA-ScoT PCR amplification system established in this paper has several advantages: simplicity,stabilization,highly reproducibility,inexpensiveness and rich polymorphism,which would be a new technique useful for sugarcane as differential gene expression analysis.