Study On A Family Member Of Frigida-Like Protein Gene AtFRL4and Its Up-Stream Regulatory Srquence In Arabidopsis Thaliana

The transition from vegetative growth to reproductive growth for plant plays an important role in its life cycle. Research on the mechanism of flowering can provid great foundation to the production of pasture and seeds, and do benificial to the adjustment of plant flowering time. Research on AIFRL4(At4g14900) gene had been performed, as well as the up-stream promoter named AtFLP. And the function and the mechanism of AtFRL4gene in flowering course were explored in this study.The whole length of AtFRL4had been isolated by PCR method from Arabidopsis thaliana according to the sequence provided in NCBI. Bioinformatics analyses were performed on the nucleotide sequence, as well as the amino acid sequence of AtFRL4. The AtFRL4cDNA is1934bp in whole length, sharing38.9%in nucleotide sequence homology to FRI.532amino acids encoded by AtFRL4gene, shared24%homology to FRI, and contained FRIGIDA multi-domain and WWbp domain. The AtFRL4protein was a hydroxyproline-rich glycoprotein homolog and belonged to the FRIGIDA-LIKE protein family.Subcellular location was performed to determine the position of AtFRL4protein. An fusion-expression vector, defined as pA7-GFP-A/FRL4, was constructed and transformed into onion epidermis cell using gene gun method. Then the AtFRL4and GFP fusion protein was transiently expressed in the control of CaMV35S promoter, and was observed by the confocal laser scanning microscope. The results showed that the fusion protein was located at nuclear with trace expression in cell membrane.In order to find the AtFRL4function, an over-expression vector called pBl-AtFRL4, was successfully constructed. Then the pBI-AtFRL4vector was transformed into Nicotiana tabacum mediated by Agrobacterium tumefaciens LBA4404. So9AtFRL4transgenic plants were obtained by kanamycin resistance screening and PCR identification on DNA, RNA level and were used to statisical test on flowering time. The statisical test demonstrated that the AtFRL4transgenic plants delayed flowering, possibly meaning AtFRL4involving in flowering time regulation. To explore the mechanism of AtFRL4in flowering course, the up-stream1690bp promoter sequence of AtFRL4was isolated. Bioinformatics analyses revealed many cis-acting elements, some of which possiblily involved in flowering regulation. A plant over-expression vector named pBI-AtFLP was constructed by instead CaMV35S promoter with AtFLP. The pBI-AtFLP plasmid was transformed into Nicotiana tabacum mediated by Agrobacterium tumefaciens LBA4404. And6AtFLP transgenic plants were obtained by kanamycin resistance screening and PCR identification on DNA level. GUS histochemical stain demonstrated GUS protein major expressed in floral organ, especially stilus, anther and filament; trace expressed in leaf, calyx and petal. Further, when AtFLP transgenic floral organs were dealed with low tempreture and dark, GUS histochemical stain results indicated that gene expression was decreased after low tempreture treatment but increased after dark treatment.