Map-based Cloning And Functional Analysis Of Sgl Gene For Grain Size And Qtl Qsdn-5 For Seed Dormancy In Rice (Oryza Sativa L.)

This thesis includes two parts:the first is "map-based cloning and functional analysis of SGL gene for grain shape in rice",and the other part is "map-based cloning and functional analysis of QTL qSdn-5 for seed dormancy in rice".Part one:Grain shape,a complex agronomic trait,plays an important role in determining yield and quality in rice.In this study,a mutant named short grain length(sgl)was identified among explants of tissue cultured japonica variety Kita-ake.Map-based cloning and functional analysis of sgl leaded to the understanding of genetic and molecular mechanism that caused short grain and dwarf.These results should be useful for application of dwarf and grain shape in breeding.Results are summarized as follows:1.The sgl mutant has a decreased seed length and weight,yet increased seed width.In addition,the sgl mutant exhibited dwarf phenotype due to shortened internodes.Scanning electron microscope analysis and paraffin sections images indicated such phenotypes of sgl mutant were caused by decreased cell size.2.The sgl locus was finally narrowed to a region of 94-kb on the long arm of chromosome 5 by map-based cloning.Twelve putative genes were located in the region by rice data base.We found one base deletion in the 5'-UTR in LOC_Os05g06280 in sgl.The expression level of the gene in sgl was decreased to 50%of that in the WT,and the reduction was associated with a decrease in the level of protein.This SGL protein was identified as a kinesin-like protein named SRS3.3.Real-time PCR was performed to study the expression profile of SGL.The result showed that SGL was expressed in various organs,including culms,flag leaves,young panicles at the booting stage and in roots at the seedling stage,and significantly higher levels of expression occurred in the culms and young panicles.The ubiquitous expression pattern of SGL is consistent with the multiple phenotype of the sgl mutant having defects in stem,spike and seed development.In onion epidermal cells,SGL protein mainly functioned in the nucleus,was also detected in other parts of cells.4.The mutant sgl can respond to exogenous GA3 to rescue the dwarf phenotype,and that sgl may be deficient in active GA.Expression analysis of genes involved in synthesis of gibberellins and their responses in sgl mutant was undertaken by means of real-time PCR experiments.The results showed that expression of the upstream genes(OsKO1 and OsKO2)was greatly decreased,while the downstream genes(GA20ox1,GA20ox3,GA3ox2)was upregulated in sgl.5.Motif analysis of the SGL protein revealed a Leu zipper motif,characterized by conserved Leu residues in the bZIP transcription factors.SGL has transactivation activity in a yeast two-hybrid system.SGL has a bZIP motif on the C terminus and bZIP proteins were proposed to bind at the site of RF2a,CCA(N)nTGG Alignment analysis revealed that the region of the OsKO1 and OsK02 promoters shared a homologous sequence,K2.Using the well-established transient expression assay of Nicotiana benthamiana leaves,we found that SGL may activate GUS expression by directly binding to K2.These data suggested that SGL protein may be involved in regulating GA synthesis and responses to affect grain length and plant height.Part two:Seed dormancy is association with preharvest sprouting,which can damage seed quality and yield in rice.Seed dormancy is a complicated quantitative trait and controlled by many quantitative trait locus(QTLs).Fine mapping and cloning of QTLs for seed dormancy not only helps us in illuminating the molecular mechanism of seed dormancy,but also improving breeding efficiency using marker-assisted selection(MAS).In this study,we did further research to fine map and clone seed dormancy QTL qSdn-5 based on the results previously.These will provide useful information to reveal the genetic mechanism for seed dormancy.1.We verified the results previously and conducted further fine mapping.The QTL qSdn-5 was finally narrowed to a region of 50 kb between two InDel markers,D9 and D7 on the short arm of chromosome 5 by map-based cloning.This region contains eight ORFs.The encoding sequences of ORF3,ORF6 and ORF8 were different between N22 and NJ35,so these genes were used as the candidate genes for further transgenic analysis.2.To determine whether ORF3,ORF6 or ORF8 underlies the QTL qSdn-5,we construced overexpression transgenic rice lines in japonica cultivar NJ35,and examined their seed germination.The results from the T1,T2 and T3 progeny seeds indicated that overexpression of ORF6 could significantly strengthen seed dormancy of NJ35.In addition,we construced RNAi transgenic rice lines in N22 and NIL5 to validate function of ORF6,and T0 Transgenic seedlings were growing in the greenhouse.3.Real-time PCR was performed to study the expression profile of ORF6.The result showed that ORF6 was constitutively expressed in various organs and particularly the highest in seeds.The expression level was increased in developing seeds.In rice protoplast qSdn-5 was located on Golgi.4.The enzyme activity was detected using expressed proteins of qSdn-5 from NIL5 and NJ35 in vitro.The results showed the enzyme activity of qSdn-5 in NIL5 was higher than that in NJ35,suggesting the difference for seed dormancy may be caused by the difference of the enzyme activity between NIL5 and NJ35.5.We measured the concentration of H2O2,a common ROS,in seeds of NIL5,NJ35 and overexpression transgenic lines.The result showed that the seeds of NJ35 contained much higher H2O2 concentration than that in NIL5 and overexpression transgenic lines.Further,we measured the alpha-amylase activity in NIL5 and NJ35 seeds.The results showed that the alpha-amylase activity in seeds of NJ35 was higher than that in NIL5 seeds,and this corresponds with stronger seed dormancy in NIL5 than NJ35.In order to explore the relationship between ABA and qSdn-5 in regulating seed dormancy,ABA content of seeds in NIL5 and NJ35 were measured by TSQ LC/MS/MS analysis.The result showed that ABA content of seeds in NIL5 was slightly lower than that in NJ35,which was not consistent with stronger seed dormancy in NIL5 than NJ35.However,NIL5 dosplayed more sensitivity to ABA than NJ35.In addition,the expression of OsDOG1L-l and OsDOG1L-2 in NIL5 was higher than that in NJ3 5,suggesting that qSdn-5 regulates potential regulators of seed dormancy.