Construction Of Yeast Two-hybrid Bait Plasmid With PPO Gene And CDNA Library From Salvia Miltiorrhiza Bunge

Due to its remarkable curative effect in the treatment of coronary heart disease, angina,myocardial infarction and cerebral vascular disease, Salvia miltiorrhiza have been widely used in clinical treatment. In recent years, it has been made into health care products and as a result, the demand is greatly increased. However, the growth of S. miltiorrhiza is easily affected by drought, nutrient deficiency, pests and other factors, resulting in lower yields.Studies have shown that Polyphenol oxidase involved in the defense responses to drought stress of plant. The expression of PPO, an important foundation for broad-spectrum resistance to plant-derived substances, can improve the resistance of plants. However, the mechanism is not clear. In order to understand the role of polyphenol oxidase in the process of resistance of S. miltiorrhiza, we screening protein and gene that interacting with PPO by the yeast two-hybrid seystem. The results are as follows.1 Full fragment of ORF of polyphenol oxidase cDNA from hairy root of S. miltiorrhiza was amplified using RT-PCR and directly ligated to the pGBKT7 vector after it was digested with NdeI and EcoRI. Insert-contained plasmid was confirmed by restriction end onuclease analysis and DNA sequencing. The self-activation and toxicity of the bait plasmid transformed into the yeast cell AH109 with empty vetor, respectively. The result was observed in SD selective medium, showing that the pGBKT7-PPO encoded by the bait plasmid could not activate transcription of the reporter genes and was nontoxic to the cells.2 A full-length cDNA library was constructed with the seedings of S. miltiorrhiza by SMART(switching mechanism at 5’end of RNA transcript) technique synthesized double strand cDNA and ligated ds cDNA fragments to pGADT7 vector. The quality of library construction was iniitially identified according to the requirements. The titer of the amplified library was estimated as 6.9×1010 cfu/ml and the recombination rate was approximately93.75%. PCR results showed that the inserts varied from 0.5 to 2.5 kb with an average size of1.5 kb or so. It indicated that this library could be used for full-length genes screening and cloning of low abundance genes.