Studies On Biological Functions Of Replication Factor C In Arabidopsis Thaliana L.

Replication factor C is a protein complex relatively conservative in many species, and it has a combination of five protein subunits, RFC1, RFC2, RFC3, RFC4, and RFC5. Studies in single-celled organism yeast demonstrate it involved in DNA replication, repair and cell cycle checkpoint control, but RFC function in plants has not to be revealed, in particular, its function in plant reproductive development. In this study, by using a series of molecular and cell biology techniques on Arabidopsis research material, we initially clarified the molecular mechanisms of DNA repair of the AtRFCl involved during meiosis. In addition, the roles of AtRFC2, AtRFC3, AtRFC4, and AtRFC5genes in Arabidopsis embryo development were investigated preliminarily. Our main results are as follows:1. We conducted a bioinformatics analysis for Arabidopsis five RFCs. Multiple sequence alignment shows that they contain eight conserved domains, which is consistent with other species of RFCs. However, the N-terminal of the AtRFC1protein contains a unique structural domain, the BRCT domain. Sequence alignment and homology-model reveal that it is very similar with other species BRCT domain. Therefore, we got AtRFCl T-DNA insertion mutant from the Arabidopsis mutant library (http://www.arabidopsis.org/) and named it rfc1-2. After mutant identification, we found that the fertility of the rfcl-2homozygous plants was significantly reduced. Further observation, we found that male and female gametophyte development has a significant defect. Among them, the mutant microspores are difficult to polarize, resulting in the subsequent mitosis disorder, and ultimately pollen sterility. As for the female gametophyte, we found that most of rfcl-2embryo sac arrested at the functional-megaspore stage, it was difficult to differentiate into functional mature embryo sac. DAPI staining of the pollen nucleus and the nucleus of somatic cell petals suggested that pollen mitosis was abnormal, whereas somatic cell mitotic morphology was normal. These results demonstrate both male and female gametophyte development were impaired.2. Meiosis chromosome morphological characteristics were observed by DAPI staining of chromosomes of pollen mother cells, the results showed that rfcl-2mutant meiosis was disrupted. Although chromosome is normal in leptotene, the early zygotene and pachytene, diplotene chromosome appeared some double-strand breaks, the rupture led to the subsequent defects of chromosome condensation and homologous chromosome separation. Although meiotic cytokinesis was not affected, still give rise to tetrads, but the genetic material of the nucleus of each microspore is impaired. The above results demonstrated that male meiosis during meiosis prophase I was affected and this defects should be the main reason or rfc1-2sterility.3. By using mRNA in situ hybridization technique, we detected AtRFC1transcription in various organs; the results showed that AtRFCl displayed abundant expression in the inflorescence meristem, and the male and female organs, especially in the microspore and megaspore mother cells. In addition, immunofluorescence technique was adopted to detect the AtRFCl signal distribution in the meiotic chromosomes. The results demonstrated that AtRFC1expressed mainly on reproductive tissues. Additionally, the high expression level in pollen mother cell was consistent with its function during meiosis.4. By hybridization, we obtained the atspoll-1rfc1-2and atrad51rfc1-2double mutants. Homozygous offspring analysis showed that the double mutant phenotypes were consistent with the single atspoll-1and atrad51mutants:both atspoll-1rfc1-2and atspoll-1displays ten univalents during metaphase I; many DNA fragmentations appeared in atrad51rfcl-2and atrad51during anaphase and telephase. The results indicated that AtRFCl should be function downstream of AtSPOll-1and work with AtRAD51during homologous recombination.5. In addition, we got the mutants of AtRFC2, AtRFC3, AtRFC4, and AtRFC5genes, the mutant identification and analysis indicated that these four mutants were the embryonic homozygous lethal phenotypes. Examination of ovules showed that mutant embryos arrested in the two-celled stage of embryo proper. Additionally, mutant endosperm cell division was severely suppressed, the size of endosperm nucleus increased, and their number decreased. By transgenic technique, we mainly analysed the AtRFC4expression pattern and its interaction with other subunits. The result showed that AtRFC4expressed highly in tissues undergoing active cell division, such as seedling, guard cell, root tip and lateral root tip. Meanwhile, in situ hybridization showed that the early embryo has its transcript. Yeast two hybrid showed that AtRFC4could interect with the other four subunits of RFC complex. The above results indicated that AtRFC4is necessary for Arabidopsis growth and development.