The Study On Cloning Of The Ribosome-Inactivating Proteins (RIPs) Gene And Its Transformation Into Sugar Beet(Beta Vulgaris L.)

Sugar beet(Beta vulgaris L.) is an important sugar crop in the world, but its yield and quality are attacked severely by many diseases. Transformation of foreign gene into sugar beet is highly efficiency in altering its characters. But little success has been got because of its low frequency of transformation. Agrobacterium -mediated transformation is the preferably approach in transgenic research due to its large transformed segments, less copies and stability of inheritance. Some researches have shown that the RIPs has the high resistant to bacteria, fungal, virus et al.In this study, maize RIPs gene has been cloned, recombined plasmid has been constructed .On the base of plant regeneration system which had been established, we studied some conditions of beet transformation, and established the high efficient Agrobacterium-mediated genetic transformation system for sugar beet. The RIPs gene has been introduced into sugar beet mediated by Agrobactium tumefaciens, and the molecular detection on transgenic plants showed it successfully. Now the major results studied in the paper are abstracted as follows:1. The whole length RIPs gene has been cloned from maize total DNA through PCR. The fragment has a full length opening reading frame of 953 base pairs encoding 307 amino acids.The gene has 99.90% homology with CRIP nucleic acid sequence reported previously,and 100% homology with its amino acid sequence.2. Introduced by the cloning vector,RIPs gene and plasmid pCAMBIA2301 were joined, thus plant expression vector with selective marker nptII was constructed.The gene was controlled by CaMV35S promoter and Tnos terminator.3. Optimized protocols of Agrobacterium-mediated transformation for sugar beet cotyledon node and plant regeneration were developed. The results indicated that the following conditions were outstanding for the improvement of transformation efficiency of sugar beet: A. tumefaciens was cultured to logarithmic state, then centrifuged and diluted again. The concentration of OD600 0.4 of the liquid inoculation medium, 24h for pre-culture and 8 min for infection and 100μM AS in co-cultivation were recommended. The favourable time and temperature are 48h and 22℃～25℃respectively. 300 mg/L Cb restrained the growth of Agrobacterium efficiently and 125 mg/L kanamycin could be regarded as the appropriate selection-pressure.4. Transgenic sugar beets were got successfully. RIPs gene was transferred into sugar beet, and 11 transgenic seedlings showed PCR positive, the positive efficiency of cotyledon node explants is up to 28.9%.The further researches correlative to this paper are right going on.