Gene Cloning, Subcellular Localization And Protein Expression Analysis Of PCNA In Brassica Oleracea Var. Acephala

Proliferating cell nuclear antigen (PCNA), which commonly expressed in eukaryotes, has shown to play important roles in DNA replication, DNA repair and cell cycle regulation. Ornamental kale (Brassica oleracea var. acephala) is an important landscape plant during the autumn and winter because it’s great chilling tolerance and ornamental value. Here, we used self-incompatibility strain S13-bS13-b of ornamental kale as material, isolated and analysed the full-length cDNA sequence of PCNA1 and PCNA2, tested subcellular localization of PCNA1 and PCNA2 by constructing the expression vector by Gateway technique,purified the PCNA1 and PCNA2 recombinant protein as antigen and prepared the polyclonal antibody.analysed the PCNA1 and PCNA2 expression in different plant tissue and different development stage of stigma by Western blot. These studies provided many important clues for biological function of PCNA. The results are mainly as follows.1. The PCNA1 and PCNA2 genes were isolated from stigma of ornamental kale by reverse transcriptase PCR and rapid amplification of cDNA ends PCR. The full length PCNAl cDNA was 1127 bp, and contained a 126 bp 5’-untranslated region (5’-UTR), a 209 bp 3’-UTR, and a 792 bp opening reading frame, which encoding a 263 amino acids residues. Predict the protein molecular weight of PCNAl is 29.16 kDa, the isoelectric point is 4.61. The genomic PCNA2 sequence was 1092 bp, which contained a 87 bp 5’-UTR, a 213 bp 3’-UTR, and a 792 bp opening reading frame, which encoding a 263 amino acids residues. Predict the protein molecular weight of PCNA2 is 28.99 kDa, the isoelectric point is 4.55. The result of the amino acid sequence alignment between PCNAl and PCNA2 showed 96% sequence similarity with 10 amino acid differences.2. Bioinformatics analysis showed PCNAl and PCNA2 have the same conserved motif, such as putative DNA binding site, PCNA/WAF1-CIP1 protein binding site, PCNA/RFCL protein binding site, PCNA/Fen-1 protein binding site, which suggest that PCNA1 and PCNA2 belong to PCNA super family.3. PK7WGF2-PCNA1 and PK7WGF2-PCNA2 expression vectors were constructed by gateway method, and transfected into Arabidopsis protoplast by PEG-Calcium-mediated method. The results showed that Egfp-PCNA1 and Egfp-PCNA2 fusion protein mainly located in the nucleus, which suggest to have important roles in DNA replication and cell cycle.4. pET-14b-PCNAl and pET-14b-PCNA2 expression vectors were constructed by using Kpn I and BamH I restriction enzyme. recombinant PCNA1 and PCNA2 protein were induced by IPTG SDS-PAGE results showed that a band with molecular weight of 35 kDa was specific induced. Furthermore, recombinant PCNA1 and PCNA2 protein were purified with Ni-NTA resins.5. The mice were immunized by purified PCNA1 and PCNA2 proteins as antigens. The PCNA1 and PCNA2 antiserum were obtained. The result of ELISA indicated that the titer of mice No.2 is better than the others immunized by PCNA1; the titer of mice No.2 is better than the others immunized by PCNA2.6.The total protein from the stem, petal, anther, style, ovary and stigma at different development stage were extracted. Western blot analysis showed that the expression of PCNA1 is highest in style and ovary, lowest in anther. and the PCNA1 have high expression in the early stage of stigma development and gradually decreased during stigma development. The protein expression pattern of PCNA2 has similarity with PCNA1. These results indicated that PCNA1 and PCNA2 may play important roles in the early stigma development.