Map-based Cloning, Complementation And Function Study Of RFC3 In Arabidopsis Thaliana

Arabidopsis thaliana, as a model plant, has established its first place in plant genetics,molecular biology especially developmental molecular biology since 1990s. RFC(replication factor C) was originally purified from human Hela cell extraction in the end of1980s and found to be required for the synthesis of simian virus 40 DNA replication in vitro.Later on, the five subunits, one big subunit RFC140/ RFC1 and the other four smallsubunits RFC37/RFC2, RFC36/RFC3 , RFC40/RFC4 and RFC38/RFC5 were found tobe existed in human, Saccharomyces cerecvisiae and many other eukaryotes. Theimportant role of RFC as a whole in DNA replication and repairing has been graduallyunderstood by people. But the study of RFC in plants progressed comparative slowly. It wasuntil 2003 that Furukawa successfully cloned the five subunit in rice using homology cloningwhich include OsRFC1, OsRFC2, OsRFC3, OsRFC4 and OsRFC5. Nevertheless, it wasconfined to molecular characterization and expression pattern study because of the lack ofthe mutant plants and thus cannot carry out function study. The reason is that RFC is anessential factor for DNA replication and the mutation in this gene must cause lethality ofthe plants. Although the sequences of the whole genome of Arabidopsis were published atthe end of 2000, there is no data about function of Arabidopsis RFC reported in the worlduntil now.As a result, in nowadays functional genomics era, it is very important to study furtherthe function of RFC, especially the molecular characteristics and biological function of eachsubunit in order to understand how its functions in growth and development as well as stressresistance from tissue, cell, molecular and also evolutionary levels, and thus understandultimately the complicate biological process integrity then establish the reference system toreinforce the recognition and understanding of relative information acquired from other plantspecies. AtRFC3 was mutagenized by EMS (ethylmethane sulfonate) and map-based cloningusing Columbia ecotype as material in this paper. Parallel study between rfc3-1 mutant andCol wild type plants were conducted to investigate the function of AtRFC3 in growth and mechanism of AtRFC3 in cell proliferation and resistance to pathogen and UV radiation werealso discussed and thus provided further evidence of the importance of the gene.The main results are as the follows:1. rfc3-1 is a recessive mutant controlled by one single gene mutation. Comparingwith wild type plants, the mutant has smaller size of configuration and narrower leaves withjagged edges. The mutation site is located within the second exon of RFC3, in which onenucleotide is mutagenized from G to A by EMS. The amino acid sequence of AtRFC3 hasvery higher identity and similarity with the homologous sequences in rice, human, mouse,African clawed frog, zebrafish, Drosophila and Saccharomyces cerecvisiae.2. The wild RFC3 gene was ligated into pG229 vector to make the reconstructedconstruct pG229-RFC3 using molecular cloning technology and the positive clone wasconfirmed by restrict enzyme digestion and further by sequencing. We got 37complementary individuals in total by agrobacterium transformation and Basta screen,whose phenotype is similar to the wild type plants. The results of the specific primer PCRshow that 8 of them have the genotype of rfc3-1/rfc3-1::pG229RFC3, which indicate thatthe mutant phenotype is reverted by the wild type RFC3 gene transformation and thus themutant phenotype is caused by point mutation of RFC3.3. The cotyledon and the first 2 pairs of true leaves of rfc3-1 mutant are smaller thanthat of wild type Col and thus the whole area of leaves is significantly smaller than wildtype. The leaf edges of the mutant are more jagged than that of Col wild type. Furtherstudies show that the epidermal cells both in cotyledons and true leaves are much biggerthan that of the wild type plants, which indicates that mutation in RFC3 gene causes somedefects in cell proliferation and thus produce less number of cells and smaller size oforgans and plant. This confirmes further the role of RFC in the regulation of cellproliferation and is also the first experimental evidence of the function of RFC in plant.4. Mutation in RFC3 gene causes the smaller configuration size of the rfc3-1homozygote and the inflorescence become short and smaller with smaller flowers andnarrower petals and thus smaller petal area comparing with wild type Col. The length ofmature silique of rfc3-1 homozygote is shorter than that of the wild type plants with 30less number of seeds per silique but 2.7 times higher number of bad seeds. It is becausethe mutation causes some defects in cell proliferation and thus produces less number of cells and leads to partial sterile in rfc3-1 homozygote.5. The average length of root in rfc3-1 homozygote is slightly longer than that ofthe wild type plants and the elongation rate in early seedling stage is higher or equal tothat of the wild type Col. This is because although the cell productivity rate of the wildtype plant is higher than that of rfc3-1 homozygote and thus rfc3-1 homozygote hassmaller number of cortex cell in the mature area of root, the length of cortex cell,however, in rfc3-1 homozygote is about 2 times the length of the wild type plants, whichcompensate the cell number defects and result in the similar elongation rate in rfc3-1homozygote.6. The mutation in RFC3 gene leads to the up-regulation of PR genes and moreresistance to pathogen Peronospora parasitica (P.p.) Noco2. At the same time, mutation inrfc3-1 compromises the resistance to UV radiation and the recovery capabilities of the 4 dayseedlings.