Gene Regulation And Effectors On Lycopene Metabolism In Carrot (Daucus Carota L.)

It becomes very imminent to improve lycopene content in plant and to make lycopene production industrialized with the special functions of lycopene on human health discovered recently, and the gene regulation and effectors on lycopene metabolism in carrot (Daucus carota L.) were studied in this experiment.The main results of gene regulation on lycopene metabolism by anti-sense RNA of lycopene b-cyclase (LYC-b) are as follows:On the basis of anti-sense RNA technology, plant expression vectors with anti-sense 306bp at 3' end, 397bp at 5' end and 739bp in the middle of lycopene b-cyclase cDNA from carrot were constructed by using CaMV 35S as a promoter, and named pBIL-3, pBIL-5 and pBIL-M. respectively.Three vectors were transformed into Agrobacterium tumefaciens GV3101 by tri-parent hybridization. In order to certify the vectors integrity during the transformation, and the GV3101 strains with different vectors were used to transform two tobacco varieties (SRI and Xanthi) by infecting tobacco leaf plates. Southern blot and lycopene analysis of leaves from transformed tobacco plants showed that the transformed genes had not only integrated into tobacco genome but also expressed.Two carrot varieties were transformed by infecting hypocotyl with the GV3101 strains checked by tobacco transformation respectively. Southern blot demonstrated the integration of anti-sense gene fragments into carrot genome, and Northern dot showed that the transformed gene had transcripted out anti-sense RNA special to internal LYC-b in carrot, and the analysis of lycopene content indicated that the expression of anti-sense RNA suppressed the expression of internal Z7C-6,which led to the contents of lycopene in transgenic plants higher than the contents in untransgenic plants. The comparison of lycopene contents among transgenic plants, callus and suspension cells with pBIL-3 pBIL-5 and pBIL-M exhibited that the influences of different fragments on lycopene content were not identical. The effect of the anti-sense of 3' end on lycopene content was the strongest, that of 5' end was the weakest, and the middle fragment was in the middle.The influences and the action mechanisms of culture condition, medium, sucrose concentration, plant growth regulators and inorganic ions on lycopene synthesis in suspension systems were studied by employing TEBE and Xin Touxinhong suspension systems as materials and two-stage culture as a method. The results are as follows:The effects of culture conditions on two cell systems were different. Lighting and proper low temperature (20 癈) were favorite to lycopene synthesis in Xin Touxinhong, whereas darkness was good for lycopene synthesis in TEBE , and temperature had no significant influence on lycopene synthesis in TEBE. The maxium value of lycopene content in Xin Touxinhong appeared during 25~30 days after inoculation, and that in TEBE appeared about 15 days.The tendence of influences of medium and sucrose concentration on lycopene synthesis in two cell systems was same. Both MG$ medium and high sucrose concentration enhanced lycopene synthesis in cells of TEBE and Xin Touxinhong suspensions.The actions of plant growth regulators on lycopene synthesis in two cell lines were not same. 3.0mg/L NAA and l.Omg/L 2,4-D improved the lycopene synthesis in Xin Touxinhong, while NAA had no significant effect, and 5.0mg/L 2,4-D significantly improved synthesis in TEBE system.Inorganic ions had different effects on lycopene synthesis in two lines. 25mM NOs" and more than lOmM P04 " most significantly improved lycopene synthesis in Xin Touxinhong , and more than 54mM NOs" enhanced lycopene synthesis in TEBE, PC>43~ had no most significant effect on it.The action mechanisms of various factors for two cell systems were different. Northern dot of mRNAs of PSY, ZDS, LYC-b indicated that the improvement oflycopene synthesis in TEBE was mainly through improving the expressions of up-stream genes including PSY and ZDS, and the enhancement of lycop...