Optimization of cDNA-AFLP reaction system for Carthamus tinctorius / 中草药

ABSTRACT

Objective To construct for the heredity linkage map and to study the functional gene research of Carthamus tinctorius,the factors affecting cDNA amplified fragment length polymorphism(cDNA-AFLP) of C.tinctorius were investigated with developing and optimizing the cDNA-AFLP reaction system.Methods Improved Trizol method was used to extract RNA from compounds in new petals specific to safflower.With the help of M-MLV RTase without RNase activity combined with replacement synthesis method,double-stranded cDNA was synthesized from total RNA;cDNA was digested by restriction enzyme MseI/EcoRI and ligated by two steps.Then the products were provided for pre-amplification and selected amplification of different concentration gradients.After tiny modifications of system concentration,finally PAGE electrophoresis and silver-staining were performed.Results High purity and integrated total RNA for later cDNA synthesis were obtained and high quality cDNA was synthesized with the help of M-MLV RTase without RNase activity combined with replacement synthesis method.The cDNA-AFLP reaction system in C.tinctorius was as follows 250 ng integrity cDNA was digested thoroughly at 37 ℃ for 6 h,and ligated 12 h at 16 ℃.Furthermore,the sample dilution multiplication was 10 fold for pre-amplification and 150 fold for selected amplification under the proper system concentration.According to the above reaction system,the polymorphous strips with high resolution power in PAGE electrophoresis were clear and stable.Conclusion The cDNA-AFLP reaction system established and optimized in this experiment is suitable for the functional gene analysis of C.tinctorius.