Studies Of Regulatory Effect Of PCNA And RFC Subunit4on Growth And Development Of Arabidopsis Thaliana

DNA replication and repair is regulated by many pathways in Eukaryotic organisms. Both proliferating cell nuclear antigen (PCNA) and Relication Factor C (RFC) complex play roles in DNA replication and repair process, two PCNA genes were discovered in Arabidopsis, PCNA1and PCNA2, but their molecular functional mechanism is still unknown. The activity of DNA polymerase8is dependent on PCNA which coordinated with DNA polymerase a controls DNA replication and repair process. In the presence of ATP, RFC can load PCNA and DNA polymerases δ onto the template with primers, and effectively extend the replicated daughter DNA strand. The biological functions of RFC4and PCNA in plants, however, is still unclear and one of the major topics remained to be solved in botany nowadays. Thus, the mutant homozygotes of pcnal and PCNA2-OE as well as heterozygote of RFC4-OE were obtained by RNA interference or overexpression experiments. The effects of gene mutation on plant growth and development as well as resistant to DNA replication toxicant methyl methanesulfonate (MMS) of different concentrations were studied using these mutants as materials. The regulatory effects of RFC4and its interactor PCNA on growth and development were also discussed in the paper.The main results are as the follows:1. The overexpression lines of RFC4-OE with obvious phenotype was screened. Comparing with wild type plants, RFC4-OE plants have smaller stature with long and slender blades and petioles. However, RFC4-OE plants are still heterozygotes because there exits phenotypic separation among the progenies. According to the results of molecular analysis, there might be some dosage accumulation effects in RFC4-OE plants and higher accumulation of expression of the gene leads to seedling lethal. At the meantime, penal and PCNA2-OE plants with stable phenotype were screened through the generations. Comparing with wide type plants, pcnal mutant homozygote which is RNA interference mutant with down regulated expression of PCNA1shows smaller configuration with early flowering, smaller but not jagged leaves, little branches, short length of root and less lateral roots. However, PCNA2-OE mutant homozygote which is overexpression line with up regulated expression of PCNA2shows dwarf stature with early flowering, fasciate rosette leaves, short but fasciate inflorescence and more branches. It is suggested that down or up regulated of gene expression of PCNAl or PCNA2affects plant growth and development significantly.2. The suppressive effects on plant growth of wild type (Col-0) and pcnal plants increased with the concentration of MMS. However, the length of the main root of pcnal plants was significantly shorter than that of wild type plants. The suppressive effects on plant growth of wild type (Col-0) and PCNA2-OE plants also increased with the concentration of MMS. But there was no significant difference of the length of the main root between PCNA2-OE and wild type plants. In addition, overexpression of PCNA2increased the enzymatic activities of superoxide dismutase (SOD) and peroxidase (POD), and showed the enhanced resistance to oxidation and damage in PCNA2-OE plants. Real time PCR analyses were also in accordance with the results and showed no compromised but even increased resistance to toxicant of DNA replication in PCNA2-PE mutant. It is indicated that AtPCNA1and AtPCNA2play an important regulatory role in DNA replication damage repair and resistance to stress in Arabidopsis thaliana.3. An experimental protocol was developed for detection of the polyploidy, number and size of the cell in Arabidopsis thaliana. It was found that the ratio of tetraploid in third and fourth leaf of penal and PCNA2-OE mutant was higher than that of the corresponding leaf in the wild type plants, but no significant difference for the size of nuclears. The number of cells and polyploid of the corresponding leaf was found to be the most one in PCNA2-OE mutant. The number of cells in corresponding leaf of pcnal was significantly smaller than that of the wild type plants, suggesting that PCNA1and PCNA2play roles in cell proliferation in plants.