Screening And Analysis Of Proteins Interacting With PCNA From Ornamental Kale

Proliferating cell nuclear antigen(PCNA)has a strong activity in the division cells and mainly involved in cell cycle regulation,DNA replication,DNA repair and epigenetic modifications.The results of differential proteomics showed that PCNA content was negatively correlated with the stigma development:PCNA expressed mainly in early development and gradually decreased during stigma developent.In this study,we screened proteins interacted with PCNA by yeast two-hybrid,and obtained the full cDNA by 5'RACE cloning,analyzed gene expression by qRT-PCR.These results provided many important clues to understand biological function of PCNA.The main results were obtained as follows:(1)The full-length of PCNA1/PCNA2 of Brassica oleracea var.acephala was cloned into pSos vector respectively.And the pSos-PCNA1 and pSos-PCNA2 plasmid were tested and confirmed to be suitable for screening protein-protein interactions in the CytoTrap system.(2)The PCNA 1 could interact with PCNA 1 or PCNA2,and PCNA2 could interact with PCNA1 or PCNA2 using CytoTrap cytoplasmic yeast two-hybrid assays.(3)Stigma proteins interacting with PCNA1 or PCNA2 as bait were screened based on CytoTrap cytoplasmic yeast two-hybrid system.Clone encoding partial ESCHBP1 and PP2C21 were showed to interact with PCNA2.The full length ESCHBP1 cDNA was 1057 bp,containing a 185 bp 5' untranslated region,a 145 bp 3' untranslated region,and a 1177 bp open reading frame,which encoded a predicted 391 amino acids.the deduced amino acid sequence shared 97%identity with Brassica napus ESCHBP1.The full length PP2C21 cDNA was 1366 bp,containing a 69 bp 5' untranslated region,a 223 bp 3' untranslated region,and a 1074 bp open reading frame,which encoded a predicted 357 amino acids.the deduced amino acid sequence shared 99%identity with Brassica napus PP2C21.(4)The qRT-PCR results showed that Tub-a6 and Actin were the most suitable reference genes among the given set of tissues and during stigma development,respectively.Furthermore,the expression of PCNA1 normalized with Tub-a6 or Actin showed that PCNA1 was predominantly expressed in ovary among the given set of tissues and during stigma development PCNA1 reached its lowest level at approaching anthesis.PCNA2 has similar expression pattern to PCNAI.The expression of ESCHBP1 showed that it was predominantly expressed in petal among the given set of tissues and during stigma development increased first and then reduced.The expression of PP2C21 showed that it was predominantly expressed in stigma among the given set of tissues and the expression keeping increased when stigma development.(5)The full-length of ESCHBP1 and PP2C21 were cloned into pMyr yeast expression vector.However,the full-length ESCHBP1 and PP2C21 didn't show to interact with PCNA1/PCNA2 in yeast two-hybrid assays.