Cloning And Molecular Characterization Analysis Of UVR8 In Prunus Avium

Up to now,UVR8(UV Resistance Locus 8)was described as only UV-B specific signaling molecules to regulate various aspects of plant growth.UVR8 gene is widespread in higher plants.However,it was mainly focused on the study of several model species such as Arabidopsis thaliana.Although the research on plant UVR8 had been carried out,few studies had been done on sweet cherry or been reported that UV-B induced the color changing on sweet cherry fruit.In this study,a novel UVR8 full-length cDNA was obtained from 'Hong Deng'(Prunus avium)by using RT-PCR and named PacUVR8.It was analyzed and identified by bioinformatics analysis,identification of tissue specificity,subcellular localization and verification of function in Arabidopsis thaliana.The main conclusions were as follows:1.A novel UVR8 full-length cDNA was obtained from Prunus avium by homology-based cloning.The results showed that the full-length gene of open reading frame(ORF)was 1 329 bp in size and encoded 442 amino acids residues(Mw=48.487 kD)and was accepted by GenBank(KX671127)named PacUVR8.Conservative amino acids domain analysis showed that PacUVR8 protein contains seven RCC1 domain structure.Homology analysis of the deduced amino acids indicated that the sequence of Prunus avium had 78%?98%identity with others UVR8 gene.2.The expression of PacUVR8 was analyzed during time of the cherry development in the tow cherries variety with the different colors by Real-time RT-PCR.The results showed that for 'Hongdeng' the expression level of PacUVR8 gradually increased along with the maturity of fruit;However 'Sato Nishiki' gene expression level was first increased and then decreased,in the color turning period of 20 days having the highest expression level.This was suggested that there was a close relationship between the change of peel color and the expression of PacUVR8 in Prunus avium.At the same time,in this study,it was also analyzed that was the expression of different tissues and organs in Prunus avium and the expression level of young stem and fruit was the highest.It was suggested that the young stems and fruits were likely to be primary organ feeling UV-B.3.Subcellular localization analysis in onion epidermal cell indicated that:PacUVR8 was localized in the cytoplasm and was dimer form.The UVR8 of dimer translate into monomer form under UV-B light;Then UVR8 of monomeroccurs interactions with downstream gene COPland recombinant protein was accumulated in nucleus.4.Verification of PacUVR8 function in Arabidopsis thaliana indicated that:over-expression vector with 35s promoter was constructed by cloned PacUVR8 gene connected to plant over expression vector.Then the recombinant plasmid vector was transferred into the wild type of Arabidopsis thaliana,and identified three over expression lines.In this study,the research results was that the Prunus avium UV-B receptor gene PacUVR8 was cloned and the function was identified.This research laid the foundation for molecular mechanism of photoreceptor response and color changing in Prunus avium.