Cloning And Functional Analysis Of Grain Size Gene DGS1 And White Stripe Leaf Gene WSL9 In Rice (Oryza Sativa L.)

This project consists of two parts:the first part is "preliminary functional analysis of grain size gene DGS1(Decreased Grain Size1)in rice",the second is "map-based cloning and functional analysis of white stripe leaf gene WSL9(White Stripe leaf9)in rice".Part 1:Grain size plays an important role in rice yield.Based on the genetic mapping by our lab,we further cloned and verified the DGS1 gene,which controls the grain size.We clarified the mechanism of DGS1 in regulating rice grain size preliminarily,which provided theoretical basis for molecular genetic basis of rice grain size and genetic improvement of rice yield.The main results are as follows:Compared with wild type Nanjing 35(NJ35),dgs1 mutant displayed distinct plant architecture with reduced plant height and increased tiller number.We performed scanning electron microscopy(SEM)on the epidermal cells of the spikelet hulls of wild type and dgs1 plants,the size of the epidermal cells of the spikelet hulls in dgs1 were obviously decreased from those in wild-type.F2 population obtained by crossing mutant dgs1 with parent NJ35 was used for genetic analysis,The results indicated that DGS1 belongs to recessive mutation.The DGS1 locus was mapped to a 140 kb region on chromosome 3 by our lab before.This region contains 16 open reading frames.Compared with wild type,there was a deletion of about 18 kb fragment in this region in dgs1 plants,which contains three open reading frames(ORFs)including Partial promoter sequence of LOC_Os03g49880,the coding region sequence of LOC_Os03g49890 and C-terminal of LOC Os03g49900.On this basis,we performed transgenic assay to verify these three genes.LOC_Os03g49880 encodes a reported TCP family transcription factor TB1 which functions as a negative regulator for lateral branching in rice.OsTB1 CRISPR transgenic plants showed significantly increased tiller number and reduced plant height compared with nipponbare.However,the grain size of tb1 CRISPR lines had no difference with nipponbare.LOC_Os03g49890 encodes a hypothetical protein(HP),HP CRISPR transgenic lines exhibitd no difference with nipponbare neither in grain size or plant height.LOC_Os03g49900 encodes a C3HC4 RING type protein.Then we used CRISPR/Cas9 to knock-out LOC_Os03g49900,transgenic positive plants all exhibited decreased grain size and decreased plant height.Thus,LOC_Os03g49890 may be involved in regulating grain size.To confirm that LOC_Os03g49900 was responsible for the dgs1 mutant phenotype we performed a complementation analysis by transforming the dgs1 mutant with the DGS1 allele.These results confirmed that the mutation of LOC_Os03g49900 resulted in the decreased grain size in dgs1.The total length of DGS1(LOC_Os03g49900)CDS is 1422 bp,and it encodes a protein containing 474 amino acids.The N-terminal of DGS1 is composed of seven transmembrane domains,and the C-terminal is a typical C3HC4 RING domain.We found that DGS1 protein is very conservative in other species such as in A.rabidopsis,Zea mays.Quantitative RT-PCR analysis revealed that DGS1 can be detected in various plant tissues,including the panicle,root,leaf,stem and leaf sheath.Subcellular localization indicated that DGS1 is localized to the plasma membrane.Putative OsBZR1 binding sites were found upstream of the DGS1 transcription start site.OSBZR1 is not only a key transcription factor in BR pathway but also involved in other growth process.Transcriptional activity assay and gel electrophoresis mobility shift assay demonstrate that OsBZR1 can bind to the promoter of DGS1 and changed the expression of DGS1 preliminarily.Part 2:Chloroplast is the main place for photosynthesis,which is a very important factor affecting rice yield.Therefore,the study of leaf color mutants is of great significance to the application in production and to reveal the mechanism of photosynthesis.In this study,we isolated and characterized a white-striped leaf mutant wsl9,identified following ethyl methane sulfonate(EMS)mutagenesis of japonica cultivar Ninggeng 3.Through genetic mapping and functional analysis,we revealed the role of WSL9 in the early development of chloroplast.The main results are as follows:The leaves of the wsl9 mutant exhibited white-striped leaves up to the third-leaf stage when planted in the field,mutant plants become green from the fourth leaf stage.Chlorophyll and carotenoid contents were reduced in wsl9 mutant seedlings.We compared the ultrastructures of chloroplasts between white sectors of wsl9 mutant leaves and wild type by transmission electron microscopy(TEM).wsl9 mutant had no organized lamellar structures and dense grana stacks.it indicated that the early development of the mutant was abnormal.The wsl9 mutant was sensitive to temperature,The phenotype of wsl9 is more obvious at low temperature.Genetic analyses indicated that the mutant phenotype was due to a single recessive allele.We isolated the WSL9 allele by map-based cloning.The WSL9 locus was initially located to a 89 kb region between InDels N12 and N3-11 on chromosome 3 in an F2 population from cross wsl9 × 9311.Sixteen open reading frames(ORFs)were predicted in the region.We sequenced the entire region in normal and mutant plants and found a SNP(G to T)in ORF LOC_Os03g07370,this SNP caused a cysteine in 128 aa to phenylalanine substitution.Complementation analysis confirmed that LOC_Os03g07370 was the WSL9 gene that caused the phenotype of wsl9 Mutant.WSL9 encodes an uncharacterized protein containing an HNH(His-Asn-HIs)domain.BLAST searches found that WSL9 protein is very conservative in other species such as in Sorghum bicolor,Zea mays.qRT-PCR indicated that WSL9 was expressed in various organs including the young leaves,roots,stems,sheaths,panicles and old leaves.Yeast two-hybrid assays indicated that WSL9 interact with MORFs and WSL which encodes a PPR protein.At the same time,we check the RNA splicing and RNA editing of wsl9 mutant.The intron in rpl2 in the wsl9 mutant was spliced at very low efficiency especially under low temperature.23 S and 16S rRNAs were significantly decreased in wsl9 seedlings grown under low temperature.Thus,wsl9 mutant was defective in plastidic ribosome biogenesis under low temperature conditions.