Gene Cloning And Preliminary Functional Analysis Of FEM6,a DNA Methylation Independent Key Regulator Of Gene Silencing In Rice

Rice is one of three main staple food crops in the world,and feeds more than half of the world’s population.With an increasing world population,the demand for rice has increased significantly.Cultivating high-yield rice varieties is an important support for ensuring national food security.Transgenic breeding,as a modern biotechnology,is a key technology for rapid cultivation of new crop varieties.However,transgenes often encounter gene silencing,which hinders the safe use of transgenic technology.In order to study the molecular mechanism of gene silencing in rice,we transformed 35S::OsGA2oxl into Taipei309(TP309),a japonica variety,and obtained a genetically modified material OsGA2ox1-overexpression(GAOE)with dwarfism,dark green leaves and late flowering.In the offspring of GAOE,an individual plant exhibited same morphology as the wild type,which was named as OsGA2ox1-silencing(GAS).GAS seeds were mutagenized with ethyl methane sulfonate(EMS),and two OsGA2ox1 overexpression mutants with gibberellin-deficient phenotypes were isolated in M2 generation and named as five elements mountain 6(fem6).This study completed the map-based cloning and preliminary functional analysis of FEM6.The results are as follows:1.Like GAOE,two fem6 mutant plants exhibited a series of typical phenotypes with gibberellin deficiency such as short plant height,dark green leaves and late flowering.Besides the morphological phenotypes,two fem6 mutants were GUS stained positively and hygromycin-resistant.Semi-quantitative RT-PCR experiments demonstrated that the transcriptional levels of GUS and HPTⅡwere recovered to GAOE,this indicates that two reporter genes also returned overexpression.2.Map-based cloning finally located candidate gene of fem6-1 into a 800 kb region near the centromere of chromosome 1.Sequencing identified that the first base of the 8th intron near the 3’ end of the 8th exon of LOC_Os01g36840 mutated G to A.This mutation caused alternative splicing,resulting in 18 base deletion at the 3’ end of 8th exon.Consequently,6 amino acids were omitted on the conserved motif(motif 10).An independent mapping of fem6-2 was also delimited the candidate gene into a same region.Sequencing identified that the last base of the 11th intron near the 5’ end of the 12th exon on LOC_Os01g36840 mutated from G to A.This mutation caused the cleavage site shift to the 12th exon,and the alternative transcript lost 34 bases,causing frame shift.The mutation started at the 440th amino acid and stopped the translation at the 456th amino acid,resulting in deletion of the conserved S5 fold domain and subsequent amino acid sequence.Using the endogenous promoter to drive LOC_Os01g36840 cDNA,the genetic complementary experiment demonstrated that plant height and transcriptional level of OsGA2ox1 was restored to the wild-type level.Allelic test between the two mutants revealed that fem6-1 is allelic to fem6-2.Genetic complementation and allelic test confirmed that FEM6 is LOC_Os01g36840.Protein phylogenetic analysis revealed that FEM6 is highly homologous to AtMORC6 in Arabidopsis.3.Despite the fem6 mutants had a significantly reduction in secondary branches number per panicle and the seed setting rate,but pollen viability,pistil morphology,effective tillers per plant,primary branch number per panicle,seed size and thousand-grain weight in fem6 mutant were comparable to that of the wild type.The performance of fem6 mutants is much better than other fem mutants,suggesting that the fem6 mutant may be an elite germplasm to prevent gene silencing.4.Chop-PCR assay revealed that the fem6 mutant had the same DNA methylation level as GAS at multiple endogenous sites,indicating that FEM6 has no effect on genomic DNA methylation level.The FISH experiment revealed that the chromatin of 45S rDNA of the fem6 mutants was significantly looser than that of the wild type,indicating that FEM6 has a regulatory role on the super structure of chromatin.In summary,this study cloned FEM6,a key factor in gene silencing.FEM6 functions in gene silencing independent of DNA methylation but by regulating chromatin structure.