Phenotype Of RFC3 Mutation And Overexpression Lines In Arabidopsis

In the life history of angiosperms,the sporophytic generation alternates with the gametophytic generation,which is known as alternation of generations.Gamephytes start to form and mature when the sporophyte grows and develops at certain stage,then double fertilization is initiated after pollenation.Afterwards,zygote and the primary endosperm form through two sperm cells of pollens fused with egg cell and centeral cell of female gamephyte,respectively.Zygote undergoes a serial of cell division and differentiation to form the mature embryo.Primary endosperm goes through syncytium stage and form mature endosperm after cellularization.Endosperm finally degrades when embryo enters the torpedo stage in Arabidopsis.During the processes of embryo and endosperm development,large amount of DNA replication occur.RFC complex,a key factor of DNA replication,plays an important role in embryo development.The RFC complex consists of five non-redundant subunits,RFC1/2/3/4/5.In the research of human and yeast,RFC complex takes part in DNA replication,DNA repair and cell cycle regulation.Athough the structure and function of RFC complex have been clearly understood in yeast and human,there is few reports on the biological function of RFC complex in higher plants.In Arabidopsis,mutation of At RFC1 causes the failure of homologous recombination and DNA replication,eventually leads to the abortion of male and female gamephytes.Mutation of At RFC4 causes blocking of DNA replication and finally leads to embryo abortion.Research reveals that At RFC3 takes part in jasmonic acid response pathway.Mutation of At RFC3 enhances the resistance to disease,which suggests the subunits of RFC complex may have other functions beside forming the RFC complex.PCNA is the DNA sliding clamp of eukaryotes,which plays an important role in DNA replication.Studies in human and yeast find that PCNA takes part in DNA replication and repair.PCNA also mediates the degration of cell cycle protein Cdt1 to prevent re-replication of DNA.In higher plants,there are a few reports on PCNA function.In Arabidopsis,there are two PCNA proteins,PCNA1 and PCNA2.Due to the lack of mutants,there is few reports on the function,interaction and composition of Arabidopsis PCNA proteins.RFC3 and PCNA sliding clamp is important in DNA replication but there are few reports on their function in higher plants.Therefore,we take a study on RFC3,PCNA1and PCNA2 genes.In this study,rfc3 mutant and RFC3 gene overexpression lines were used as the mainexperimental materials.The phenotype of rfc3 mutant and At RFC3 overexpression plants were studied through bioinformatic,genetics,cellular biology and molecular biology techniques,such as promoter sequence analysis,ovule clearing,pollen stainning.Gene editing vectors of PCNA1 and PCNA2 were constructed.Transgenic lines were obtained by flower-dipping.The main results of this study were summerized as follows:1.The genome type of T-DNA insertion mutants of Arabidopsis RFC3 were identified and analysed by three-primer PCR,and only heterozygous plantlets could be obtained.Siliques in heterozygous plants of rfc3 mutant with a proportion of 24.85%abnomal white ovules,which eventually shrank and aborted.This ratio matched the ratio of recessive homozygous death,indicated the homozygous plants of rfc3 mutant may led to embryonic lethal.In order to clear the reasons of embryo abortion,the ovule in heterozygous plants of rfc3 mutant was observed via whole mount ovule clearing.The results showed that the reduction in the number of free endosperm nuclear initiated at the elongation zygote stage.Moreover,we found that all embryos of white abnormal ovule were arrested at 2/4-celled embryo stage and the number of endosperm free nuclei were significantly decreased.In one celled-embryo stage,the averange number of endosperm free nuclei of abnormal ovule decreased to 5.22,while the wild type ovule at the same stage was 17.45.These results revealed RFC3 gene mutation led to the arresting of both embryo and endosperm,and eventually to the seed abortion.2.In order to study the function of RFC3 gene,35S::Venus-RFC3 transgenic lines were obtained through transgenic technique.Four RFC3 transgenic lines were selected as materials in following research.In siliques of RFC3 transgenic lines,a part of ovules were aborted.In RFC3OE3-1,the abortion rate was 13.79%.In RFC3OE4-1 the abortion rate was 9.79%.In RFC3OE6-2,the abortion rate was 6.56%.In RFC3OE9-1,the abortion rate was 1.99%.While the abortion rate of wild type was 1.89%.It suggested that transgene of 35S::Venus-RFC3 plasmid might lead to the ovule abortion.Pollen abortion in these lines were found via pollen staining.KI-I₂ staining was used to mesure the maturity rate of pollens.The abortion rate was 12.57%in RFC3OE3-1,14.69%in RFC3OE4-1,5.90%in RFC3OE6-2,6.14%in RFC3OE9-1.These reults also suggested the transgene of 35S::Venus-RFC3 might lead to the pollen abortion.The results of whole mount ovule clearing revealed that the embryo development was faster than wild type in RFC3OE3-1 and RFC3OE4-1.The development of RFC3OE3-1 and RFC3OE4-1 embryos was similar to wild type.These results suggest,transgene of 35S::Venus-RFC3 might lead to the accelerating of embryo development.3.The PCNA protein sequences in 18 defferent spicies,including Homo sapien,Caenorhabditis elegans,Saccharomyces cerevisiae,Arabidopsis thaliana and Oryza sativa and so on were used for sequence alignment and evolutionary tree analysis.Results showed that PCNA protein sequences in all 15 species were conserved.There were 2 PCNA proteins in Arabidopsis,while there is only 1 PCNA protein in Homo sapiens and Oryza sativa,it showed that the number of PCNA protein in different species were different.The promoter analysis of PCNA1 and PCNA2 showed that there was a E2F family transcription binding site on PCNA1 promoter.And there is stress response factors on PCNA1 promoter,suggested PCNA1 might take a part in stress response.There was M phase expression factor on PCNA2 promoter,which was not found on PCNA1 promoter,suggesting that PCNA2 gene might take part in cell cycle regulation.4.PCNA1-CR,PCNA2-CR and PCNA1/2-CR gene-editing vector were constructed for flower-dipping transgenic technique,and 5 PCNA1-CR transgenic lines,9 PCNA2-CR transgenic lines and 6 PCNA1/2-CR transgenic lines were obtained.The transgenic background of these lines were identified,and the fragments contained the target site were sequenced,but no gene editing was not found on target site,which indicated the needs of further anslysis of transgenic of PCNAs.