| Lycium ruthenicum Murr.is a perennial shrub mainly distributed in the northwest of China that belonges to the family of Solanaceae,Lycium genera.Its mature fruits contains many kinds of active ingredients,such as anthocyanins and proanthocyandins,which have strong antioxidant activity,and is used to be common medicine of minority.However,there are a less of study involve in the biosynthetic pathway of these active ingredients,and the accumulation and changing of its composition in the growth process is unclear.In this study,the key enzyme genes in the anthocyanin and proanthocyandins biosynthetic pathway of Lycium ruthenicum were cloned;and the accumulation and changing of active ingredients in different growth periods was explained by the chemical composition analysis and key enzyme gene expression analysis;what’s more,the genes were successfully recombined into the viral vector,which were expressed in tobacco by instantaneous expression technology,and analysed the changing of the chemical composition.The main findings were presented as follows:1.The high fidelity polymerase and specific primers was used to amplificate the key enzyme gene(LrDFR,LrANS,LrLAR and LrANR)that contorl the biosynthetic pathway of anthocyanin and proanthocyandins in Lycium ruthenicum,whose length was 1140,1251,1002 and 1017 bp,encoding a protein of 379,416,333,and 416 amino acids,respectively.And there are characteristic sites and structure domain of the corresponding family were found in these four putative protein.The results of phylogenetic analysis revealed that all these proteins have a close relationship with the proteins from the same family,like Solanum lycopersicum,Solanum tuberosum and Nicotiana tabacum.2.Ultrasonic was used to extract anthocyanins and proanthocyandins in Lycium ruthenicum from four different growth period,following by the content dectection with ultraviolet spectrophotometric.After comparative analysis of their content changes,we found that the content of anthocyanins increase gradually as the fruits growing and developing,and the highest content was 6.08 mg/g DW when the fruit in Stage 4.However,the change trend proanthocyandins content was increase in earlier stages,then decrease slightly at mature stage,whose highest content was about 22 mg/g DW at Stage 3.3.The application of real-time quantitative PCR technology for quantitative analysis of LrDFR,LrANS,LrLAR and LrANR in four stages of fruits growing.The expression level of LrDFR and LrANS was rise with the maturity of fruit color,and the expression level of LrLAR and LrANR would be down after earily rising.What’s more,the biosynthesis of proanthocyandins was mainly controlled by LAR at the early stage of developing fruit,while ANR controled their metabolic pathways at last two stages.4.The genes of LrANS,LrLAR and LrANR was recombined into PVX viral vector-pGR107 by the digestion with double enzyme(Cla I and Sal I).Then the recombinant plasmid was transferred into tobacco for instantaneous expression with agrobacterium mediation.The results show that the content of anthocyanins and proanthocyandins in tobacco leaves which was expressed recombinant plasmid LrANS-pGUR 07 increased significantly.And the proanthocyandins content increased while anthocyanins content was not available like control groups as expressing LrLAR-pGR107 or LrANR-pGR107 in tobacco leaves.These results demonstrate the key functions of LrANS,LrLAR and LrANR in the biosynthesis pathway of anthocyanins and proanthocyandins. |
