Molecular Cloning And Functional Analysis Of Two MADS Box Genes From Peach (Prunus Persica)

Understanding the processes regulating the phase transition from vegetative to reproductive growth is particularly important for woody fruit crops. It is very likely that at least some of the genes signalling the end of the vegetative phase and the beginning of the reproductive phase are members of the MADS box gene family.MADS box genes family plays a key role in the molecular regulation of floral development, and most of them in plants belong to the typeâ…¡ class, that is, the gene sequence is composed of M, I, K and C domains. The M domain, which encodes the DNA-binding sequence and plays the roles of both binding DNA and forming dimmers, is the most conserved region. According to the classical "ABC " model, three classes of genes act to specify floral organs, namely sepals (A only) , petals (A+B) , stamens (B+C) , or carpels (C only) . Later, some new found MADS box genes result in the "ABCD" model, "ABCDE" model and "quartet" model. All of these models make the understanding of floral development deeply.Recent studies demonstrated that the functions of MADS box gene family members are not restricted to flower organ development. MADS box genes were shown to be involved in initiation of flowering, determination of meristem identity , embryonic development , root formation , development of vascular tissue, and seed and fruit formation. Furthermore, a single MADS box gene may have different functions at different stages of development.With the aim of finding genes involved in the floral transition of Prunus species (Prunus sp.), the EST (Expressed sequence tags) sequences were extracted from the public databases. Eight MADS box genes' cDNAs were obtained. Four of them were cloned from peach (Prunus persica), named PpMADS2 , PpMADS4,PpMADS6, PpMADS7.PpMADS4 and PpMADS6 have got the accession numbers in Genbank. (The accession numbers are AY705972 and AY705973). The PpMADS4 full length cDNA is 850 bp long.It contains an open reading frame of 732 bp, coding for a polypeptide of 243 amino acids . The PpMADS6 full length cDNA is 1190 bp long.It contains an open reading frame of 768 bp, coding for a polypeptide of 256 amino acids.PpMADS4 closely resembled the Arabidopsis AGAMOUS gene. It is an AGAMOUS -like C class MADS box gene and expressed in petals, carpel, fruit and nucleolus as shown by RT-PCR analysis. PpMADS6 is likely to be the peach orthologue of the Petunia PFG genes and it is an A class MADS box gene. It has been shown with RT-PCR that it expressed in sepal, petal, carpel and fruit. It may be involved in the transition from the juvenile to the adult stage.An expression vector was then constructed in which PpMADS4 gene was under the control of CaMV 35S promoter, and Arabidopsis thaliana was transformed by floral dip method mediated by Agrobacterium tumufaciens. Transgenetic Arabidopsis have been obtained by antibiotic screening and PCR screening. The flower morphology and plant configuration of the transgenic Arabidopsis were varied. This showed that the PpMADS4 gene perhaps take part in the determination of floral organ. From the above results, we proposed that PpMADS4 had the function of C class of MADS box genes.