Construction Of RNAi Expression Vectors For Tomato ARF1,ARF2 Gene And Transformation Of Tomato

Tomato(Lycopersicon esculentum) ,Which is a kind of annual herbage and belongs to the Solanaceae family in the plant kingdom. The fruit contain plentiful nutrient contents and richer sweet juice. It is one of the most important vegetables and one of the model plants which are most studied in the biology.Auxin is a kind of important phytohormone. It affects plant growth and development, including cell division and differentiation, apical dominance,plant phototropism and aging ,initiation of flowering ,floral organ abscission and et al. Auxin response factors (ARFs) are a kind of transcription factors that mediate responses to the plant hormone auxin. They play an important role in auxin signaling regulation. Therefore, we plan to obtain the transgenic tomatoes by using plant genetic engineering for seperatedly silencing ARF1,2 gene of tomato ,then do some researches on their functional analysis by observing the changes on the plant growth and development ,especially on fruit pigment.In this study, RNAi plant expression vectors with ARF1,2 were seperatedly constructed, and transferred into Ac+ tomato mediated by Agrobacterium tumefaciens. On the other hand, some factors in tomato transformation system were optimized. Using this system, transgenic tomato plant were obtained. It could be the base of the further study on the function of ARFs genes. The main results were summarized as follows:1.According to the sequence of DR12 gene, Auxin Response Factors homolog, we obtained the partial cDNA sequences of ARF1,2 genes by using Blast method. PCR primers were designed seperatedly based on both sequences. Two about 2 Kb fragments were cloned , which lay the basis for the next study on the construction of plant expression vector.2. The RNAi plant expression vectors (pBI121-ARF1,pBI121-ARF2) were constructed in the experiment. Both two vectors are controlled by promoter CaMV35S and contain the selectional label NPTII gene.3. Several important factors have been investigated on affecting Agrobacterium-mediated tomato transformation system in tomato cultivars Ac+. The results showed that the suitable concentration of NaClO for sterilization is 5%, and the seeds have no need for vernalization. The concentration of kanamycin for selection is 50mg/L in the period of resistant selection. Furthermore, It is more beneficial for root regeneration in MS + 500mg/L Carbenicillin +2mg/L IAA.4. Using this transformation system mediated by A.tumefaciens, we obtained 18 useful transgenic tomato plants including 10 arf1 and 8 arf2. these plants have been identified by PCR. And the others will be identified in the future.