Cloning And Functional Study Of Two Grain Size And Grain Number Related Genes 08sg2 And OsSpl18 In Rice

Grain size and grain number,as the direct component factors of rice yield,are significant for rice yield.In the past decades,a lot of genes related to grain size and grain number had been cloned,however,the regulatory mechanisms underlying them remains largely unkown.In this study,we identified a rice small grain(sg2)mutant in an EMS mutant library generated from an excellent indica variety,Shuhui498(R498).We investigated the agronomic traits of sg2 and made gene cloning.We obtained the main results as below:1.Compared with R498,the plant height of sg2 was reduced 19.3%,panicle length was decreased 24.3%.The grain length was reduced 20.4%,led to a 25% reduction in1000-grain weight.We generated a F2 segregation population from a cross between sg2 mutant and R498,the distribution of grain length in population suggested that two recessive genes were responsible for the sg2 phenotype.Using the Mut Map gene mapping strategy,we identified two linkage regions on chromosome 7 and 8,and named as 07sg2 and 08sg2,respectively.In this study,we focused on 08sg2.2.Three linkage SNPs with SNP index of 1 on chromosome 8 were identified,only SNP2 was located in the kinase domain of OsBAK1,and led to an amino acid substitution(Gly310Asp),the other SNPs were not located in the coding region.We isolated the 08sg2 single mutant plants using marker based on the SNP2 in OsBAK1,and 08sg2 was backcrossed with R498 three times to eliminate other potential mutations.3.Compared with R498,the plant height of 08sg2 was reduced 8%,panicle length was decreased 11.9%.The grain length and grain width was reduced 10.2% and 2.7%,respectively,led to a 12.8% reduction in 1000-grain weight.The number of primary branches had no obvious difference,but the number of secondary branches was decreased8.6%,led to a 9.7% reduction in grain number per panicle,which might be caused by the up-regulated expression of two negative regulators of panicle branch genes Os CLV1 and Os CLV2.4.Cytological analysis indicated that the cell length of 08sg2 had no obvious difference,but the cell number was reduced in the longitudinal direction.Quantitative PCR analysis showed that many cell-cycle related genes were down-regulated in 08sg2,which were involved in the G1-to-S transition.These results indicated 08SG2 regulated spikelet hull development by affecting cell proliferation.5.Knocking out OsBAK1 in the japonica Nipponbare background observed the similar phenotypes as 08sg2,which confirmed the idea that the SNP2 in OsBAK1 was responsible for the 08sg2 phenotype.But the change percentage in Nipponbare background was less than in R498 background,indicated that the function of OsBAK1 was different in various backgrounds in rice.6.A series of BR sensitivity test experiments indicated 08sg2 was insensitive to24-epi BL,consistant with the notion that 08SG2/OsBAK1 was involved in the BR signal transduction.The mutation of 08SG2/OsBAK1 led to feedback regulation of BR biosynthesis related genes,which might partially rescue the defect of BR signal transduction.7.The amino acid substitution(Gly310Asp)in 08sg2,which was located in the kinase domain of OsBAK1 and the residue was extremely conserved in plants,might lead to the protein loss the kinase activity and biological functions.Considering that the 08sg2 had a similar phenotype as the knock-out mutant of OsBAK1,we speculated that 08sg2 was a complete function loss mutant.8.Pyramiding analysis of 08sg2 and NIL-GS3R3551 at the same R498 background indicated that 08SG2/OsBAK1 and GS3 just had additive effect without interaction effect.In addition,the transcriptional levels were not affected by each other,suggesting that08SG2/OsBAK1 and GS3 regulate grain size in independent pathways.Grain size and grain number are two important traits which directly influence grain yield in rice.To date,a lot of genes related to grain size and grain number have been cloned.SPL family genes are plant-specific transcription factors,involved in diverse functions in plants.As reported,most of the SPL genes have high expression levels in panicle,and participate in regulating the panicle traits positively,including grain size,panicle branch and grain number.In addition,SPL genes usually regulate tillers number negatively.Therefore,appropriate expression levels of SPL genes facilitate to balance the tillers number and grain number,which lead to ideal plant type with reduced ineffective tillering,increased grain number and grain yield.In this study,we analyzed the SPL family genes and the OsSPL18 gave rise to our attention,which has a very similar gene structure and expression pattern with the cloned grain size related gene GW8/OsSPL16.As reported,OsSPL16 and OsSPL18 are located at the same branch of the phylogenetic tree,and they are parallel homologous gene,indicating that they maybe have similar biological function.To explore the biological function of OsSPL18 in rice,we applied reverse genetics and obtained the knockout mutants of OsSPL18 in the japonica variety Nipponbare background,subsequently,we investigated the agronomic traits comprehensively.In addition,we analyzed the expression pattern and subcellular localization of OsSPL18 and so on.We obtained the main results as below:1.We generated knockout(KO)mutants of OsSPL18 with CRISPR/Cas9 gene editing system in the japonica variety Nipponbare background,two target sites were designed,and six independent homozygous mutants with different mutation types were obtained,all mutations led to frameshift and premature stop codons.And then,we obtained the homozygous plants without carrier in T2 generation to investigate the agronomic traits comprehensively.2.All the KO lines showed a similar phenotype with each other.Compared to the wild type,the plants height had no obvious difference,but the tillers number was increased 20.3%in average;The grain width and grain thickness decreased 5.7% and 7.3%,respectively,but grain length was no obvious difference,resulting a 12.0% reduction of 1000-grain weight;In addition,the panicle length was reduced 6.0%,the primary branch number had no obvious difference,but the number of secondary branches was reduced 17.6%,resulting grain number per panicle was reduced 10.6%.Combined with these changes,the grain yield per plant was decreased 17.3% in KO lines.3.The overexpression lines of OsSPL18 in Nipponbare background exhibited reduced plant height and tillers number,accelerated flowering,additionally,the panicle size and grain number were reduced.These results indicated that OsSPL18 had pleiotropic effects,which regulated grain size and grain number positively but regulated tillers number negatively.4.Cytological analysis indicated that the cell size of KO mutants had no obvious difference,but the cell number was reduced in the transverse direction.Quantitative PCR analysis showed that many cell-cycle related genes were down-regulated in KO mutants.These results indicated OsSPL18 regulated spikelet hull development by affecting cell proliferation.5.The expression pattern of OsSPL18 in various organs was investigated by quantitative RT-PCR.OsSPL18 transcripts were detected in all the tissues observed,but the expression levels were significantly higher in developing young panicles than those in other tissues.GUS staining analysis showed that the OsSPL18 promoter had a more strong activity in developing spikelet hulls than others.Taken together,the expression pattern of OsSPL18 supported its biological function.6.The subcellular localization of OsSPL18 was located in nucleus.Transcriptional activity assay indicated that OsSPL18 had transcription activation activity,and both the N-terminal and C-terminal of OsSPL18 showed transcription activation activity in the truncated fragments test.These results indicated that OsSPL18 is a functional transcription factor with activation activity.7.We investigated the expression levels of genes related to grain size and panicle architecture by quantitative PCR analysis,as a result,the grain size related gene GW7 was up-regulated and the panicle architecture related gene DEP1 was down-regulated in KO mutants.The yeast one-hybrid experiment showed that OsSPL18 could directly bind to the promoter of GW7 and DEP1,and truncation experiment showed that the SBP domain of OsSPL18 was necessary for DNA binding.Taken together,our results suggested that OsSPL18 might regulate grain size and panicle architecture by negatively regulating GW7 but positively regulating DEP1 in rice.8.Sequence analysis indicated that OsSPL18 contains the Osmi R156 k target sequence in the third exon.RLM-RACE showed that OsSPL18 can be directly cleaved by Osmi R156 k in rice and the cleavage site was located between the twelfth and thirteenth base(from 5’ direction).Quantitative PCR analysis showed that the expression level of OsSPL18 was significant down-regulated in the Osmi R156 overexpression plants.Thus,these results indicate that OsSPL18 is the target of OsmiR156 k.