Cloning And Analysis Of Flower Development Related MADS-box Genes From Sweet Cherry (Prunus Avium L.)

Sweet cherry, which owns the high price and good benefit in the international and domestic market, is one of the favorite fruits, because of its early maturation, large fruit, attractive colour and rich nutrition. However, there are some problems such as shy fruit-bearing, fruit malformation and unsteabe yield in the cultivation practise, which mainly results from the high temperature in spring and the period of flower bud differentiation. MADS-box genes play an important role in floral and fruit development and have close relation with pollination, fertilization and the fruit development. In order to solve the problems in sweet cherry production, we attempted to isolate the genes related with flower and fruit development, and to explore the molecular mechanism caused the problems. In this study, Prunus avium L.'Lapins'was used as the material, RT-PCR and RACE approaches were used to clone the full length cDNA of MADS-box, and real-time RT-PCR was used to detect the gene expression. The main results are as follows:1. The primers in the conserved domains were designed, to get cDNA full length with RACE method, and four MADS box homologous genes were named PaMADS2, PaMADS3, PaMADS4 and PaMADS5. The four genes belonged to B(PaMADS2), C(PaMADS3, PaMADS4) and E(PaMADS5) of MADS-box gene, respectively.2. Tissue specific analysis showed that PaMADS2 expressed in petals and stamens, which was consistent with class B. Phylogenetic analysis indicated that PaMADS2 was highly homologous to PI clade of B, so PaMADS2 should be one of class B.3. Phylogenetic analysis indicated that PaMADS3 and PaMADS4 belonged to the different glades of class E. RT-PCR revealed that PaMADS3 expressed in petals, stamens and carpel, but PaMADS4 expressed in all the four wheels of floral organs.4. When shoots with flower buds were put under the different temperature(15℃/25℃) from the phase of side green to the phase of anthesis, the different genes expression patterns were observed. The expression of PaMADS3 in carpel was much higher at 25℃than at 15℃. The result suggested that the expression of PaMADS3 would be related to the response of flower development to temperature.5. One of class C gene was isolated and named PaMADS5, which expressed in stamen and carpel. Phylogenetic analysis showed that it was highly homologous to AGAMOUS glade of class C, so it should belong to clade euAG .6. The solutions of 10 mg/L, 50 mg/L , 100 mg/L and 200 mg/L GA3 were sprayed on bursting flower buds respectively, the expression of PaMADS5 in carpel was determined using real-time RT-PCR. The results indicated that at anthesis, the expression of different GA3 treatment was higher than that of control, but there were no remarkable changes among GA3 treatments; 2 days after anthesis, the expression of GA3 200 mg/L was the highest, however, the expression of 10 mg/L, 50 mg/L and 100 mg/L was lower than that of control. It was suggested that the inhibition effect of the high solutiong of GA3 on embryo sac might result from the the expression of PaMADS5.