Study On The Stability And Cloning Analysis The Key Genes Of Anthocyanin Biosynthetic Pathway In Dioscorea Alata Linn

Purple water yam(Dioscorea alata L.)has many nutrient substance and high medicinal value for health function.It is processing resource both of medicine and food.The purple water yam with broad adaptability and low waste,and the demand of its growing is not high.The turber can be used in extraction anthocyanins in industrial production,meanwhile with considerable commercial applications.Anthocyanins biosynthesis pathway have been deeply studied in the plants such as blueberry,grape and Chinese wolfberry.There is also a great limitation ahead of the research on anthocyanins biosynthesis in yam.This study mainly did extraction anthocyanins from purple water yam,and analyzing the stability of anthocyanin.Cloning the key genes in biosynthesis pathway of anthocyanins and bioinformatics analysis.Real-time quantitative PCR was used to study the critical gene express in tuber and tissues.It can be utilized in industrial production.All of these study contributed to the research and further exploration on anthocyanins biosynthesis system in purple water yam.Main contributions of this work are as follows:1.The anthocyanin compositions in purple water yam are mainly cyanidin were analyzed by HPLC.The best extraction conditions was as follows:1.5%hydrochloric acid ethanol as extracting agent,solid-liquid ratio 1:25(g/mL),time is 2.5h.The yield of anthocyanidin from the purple water yam was 67.72%on the optimum conditions through the orthogonal array design.The effect of light,temperature,pH and metal ions on the stability of anthocyanins.The results showed that the anthocyanins had good stability at the light and the temperature below 40?.In the lower pH the anthocyanins difficult decomposition.The metal ions Fe2+,Fe3+ and Cu2+ significantly influenced the stability of anthocyanins.2.The full-length DaPAL,DaCHS,DaCHI and DaUFGT genes were isolated by RT-PCR technology in purple water yam tuber.The bioinformatics analysis showed that the four proteins with typical family protein characteristics.DaPAL and DaUFGT belong to hydrophobic proteins.DaCHS and DaCHI belong to Hydrophilic protein.The amino acid sequence alignments revealed that the sequence of DaPAL shares 88%identity with Phoenix dactylifera PAL.DaCHS shares a high identity with the Elaeis guineensis homologues about 90%.DaCHI shares a high identity with the Canarium album homologues about 67%.DaUFGT shares a high identity with the Phoenix dactylifera homologues about 52%.3.Real-time PCR analysis indicated that the key genes were used to detect the expression patterns in different tissues.The expression levels of purple water yam DaCHI,DaF3 'H,DaANS and DaUFGT genes were significantly higher than white and yellow water yam.The DaF3'5'H gene had the highest expression in white water yam.The DaF3 'H gene had the highest expression in yellow water yam.The DaCHS,DaCHI,DaANS and DaUFGT genes expression exhibited an obvious increase at forming and maturity time in tuber.4.In order to study the function of DaCHS and DaCHI agaist stress tolerance.DaCHS and DaCHI proteins expressed in E.coli.The results show that the recombinant proteins enhance the stress tolerance in E.coli cell.