Construction Of DR1 Gene Plant Over-expression Vector And Research On Transformation Into Tomato

DR1 genes are new fruit ripening genes from tomato by differential display PCR, belong to auxin response gene family (Aux/IAAs gene family)..DR1 genes are regulated by auxin and ethylene,which means the genes not only are related to fruit ripening ,but also involved in the transaction between auxin and ethylene.However,what mentioned above is not clear,our research is to investigate the effect of DR1. on fruit ripening and hormone signal transduction by over-expression of DR1 in tomato,then ascertain the function of DR1.The main contents and results of the experiment as follows:1Construction of plant binary over-expression vector pBIN19-DR11)Total RNA was abstracted from mature-green tomato by guanidine thiocyanate-phenol-chloroform method;2)Total RNA as template, complement DNA of DR1 mRNA was reverse transcripted in vitro; the cDNA as template in the following PCR reaction, the whole DR1 gene was amplified by high-fidelity pfu DNA polymerase;3)Blunt-end DRl gene was ligated to pDH51 vector cut with Smal by T4 DNA ligase, transformed into competent E.coli, recombinant was screened by PCR.The recombinant was verified as positive by further PCR and enzyme cutting, named after pDH51-DRl.4)A fragment including 35S promoter, DRl gene and 35S terminator was excised from pDH51-DRl with EcoRI, the fragment was ligated to pBIN19 cut with EcoRI, transformed into competent E.coli, recombinant was screened by PCR.The recombinant was verified as positive by further PCR and enzyme cutting and sequencing, named after pBIN19-DR1.2 Gain of regenarated tomato1)pBIN19-DRl was mobilized into Agrobacterium tumefacious strain C55 by freeze-melt method, recombinant was screened by PCR. The recombinant was verified as positive by further PCR and enzyme cutting.2) AlisaCring WT tomato seeds were planted in MS substrate, germinated in aseptic condition. 0.4×0.6 cotyledon or 0.5cm hypoderm was excised as explants.3)Explants were placed in germinating substrate and precultivated for three days in dark at 28℃ temperature, then infected by recombinant Agrobacterium suspension for 30 min, co-cultivated in germinating substrate with Agrobacterium for two days at 28℃ temperature, then transferred into Agrobacterium-restrained substrate with 500mg/L Carbanicilin and cultivated for one week. Transformants were screened in 50mg/L Kan MS substrate. Transformants germinated after 40 days, then roots were induced in rootage substrate. The whole frond can be transferred into soil when roots were well-developed.