Functional Study Of OsGBP Transcription Factor Family Genes And Cloning Of A Male And Female Sterility Gene In Rice

Transcriptional regulation is an important regulatory mechanism in eukaryotic gene expression.It plays an important role in plant growth and development,stress and disease resistance response,signal transduction and secondary metabolism.Transcription factor(TF),also known as trans-acting factor,takes part in numerous biological processes and widely distributed in plant genome.GAGA-binding protein(GBP)is a kind of plant specific transcription factor that can specifically bind to GAGA repeat element to regulate downstream gene expression.In our research,rice OsGBP family was identified and analyzed within genome-wide level by bioinformatics methods,and the evolution of the family and the function of OsGBPs were also studied with transgenic method.The main results are as follows:1.Four OsGBPs homologous genes were identified in rice genome.According to the phylogenetic analysis,we found that grass GBP family members were divided into two subgroups.Further study show that the class?contain OsGBP1,whereas OsGBP2,OsGBP3 and OsGBP4 were tandem distributed on chromosome 10 of rice genome in the class?.Sequence similarity analysis showed that OsGBP1 share with the highest similarity to 53% with other members in this family and the three genes in class?share with the lowest similarity to 82% each other.In addition,OsGBP2 and OsGBP3 are two copies with the same geneome sequences.Structure analysis showed that there is a highly conserved zinc finger domain of DNA-binding in the C-terminal of all the OsGBP proteins,whereas structure difference was also observed between these two subgroups.In N-terminal of OsGBPs,OsGBP1 contains a Coiled-coil domain which mediates protein-protein interaction,but only an unknown function conserved domain was identified in other three OsGBP family members.Gene expressional data showed that OsGBP1 and OsGBP2/3 were constitutively expressed with the highly level in spikelets,whereas the expression of OsGBP4 was not detected.Subcellular localization analyses showed that OsGBP1 and OsGBP3 are located in the nucleus,and have transcriptional activation activity.In addition,the synteny analysis results reveal that OsGBP1 has strong collinearityamong barley,sorghum,maize,rice and brachypodium,which indicate its important role in evolution.2.Through the study of different genotype OsGBP1 materials(Wide type,Overexpression,RNA interference,osgbp1 mutant),we found that OsGBP1 negatively regulates grain length by affecting the expression of grain-shape related genes.Furthermore,OsGBP1 also negatively regulates seedling growth and development.Our results showed when overexpressed OsGBP1,the growth and development of seedling was inhibited,including shorten seedling height,decreasing seedling biomass.The opposite phenomenon was observed in OsGBP1 expression inhibited or osgbp1 mutant plants.In addition,we also found that the overexpression of OsGBP1 delayed rice flowering and also decreased plant height.Further studies indicated that OsGBP1 can inhibit the expression of downstream Ehd1,Hd3 a and RFT1 by up regulating the expression of flowering related genes Ghd8 and Os LFL1 to delay flowering.In vivo yeast one-hybrid and in vitro EMSA results showed that OsGBP1 directly bind to the GAGA repeat elements in Os LFL1,Ghd8 and Hd3 a promoters.Hence,we speculated that OsGBP1 regulated rice flowering was not a simple upstream and downstream relationship but a relatively complex biological process.3.We constructed OsGBP3 RNA interfernce and overexpression materials.Our results showed that OsGBP3 did not affect the growth and development of rice seedlings,but it influenced plant height and positively regulated grain length by promoting the expression of grain shape related genes.The grain length,grain width and plant height of OsGBP3 overexpression plants were significantly increased when compared to wide type,whereas they were decreased in RNA interference plants except grain width.4.To investigate whether there is functional differentiation among OsGBP family genes,we constructed double RNA interference materials of OsGBP1 and OsGBP3 on Nipponbare background.Our results showed that there is no significant difference in grain length compared with wild type whereas significant decrease in grain width.The results suggested that there are antagonistic effects of OsGBP family membersin rice grain length regulation but functional redundancy effects in grain width control.Furthermore,the plant height of double RNAi plants was significantly lower than that of OsGBP3 RNA interference alone,whereas OsGBP1 single suppression did not affect plant height.5.We obtain a 3' UTR region T-DNA insertion mutant of OsGBP1 from Korea rice mutant library.The homozygous mutant showed male and female sterility but it was not co-segregated with phenotype.Further studies showed that the gene named MFS controlled sterility and linked with T-DNA insertion.6.In order to clone the gene,we constructed a F2 population by cross mfs heterozygous mutant with wide compatibility material Dular.We succeed in fine mapping MFS to a 68 kb genomic section which contains 10 genes named GR1 to GR10.By comparing the sequencing between fertile and sterile materials,we found that there is a single site mutation in the the third gene(GR3)intron in sterile material.Further studies showed that the site mutation causes a frame shift phenomenon by abnormal splicing and lead to premature termination of GR3 protein.7.Through genetic complementation and GR3 knockout experiments,we verified that GR3 is the candidate gene of MFS,which may cause male and female sterility by affecting the meiosis of auxocyte.8.Our results showed that there are two transcripts MFS-1 and MFS-2 of MFS gene in rice and the encoded proteins of them both located in nucleus.Strangely,only the MFS-1 can interact with Os HUS1 and Os Rad 9 to form a trimeric complex.Additional knockout of other protein in this trimeric complex also leads to pollen sterility.