Screening Of An Enhancer Trap Mutant Library In Rice And Isolation And Study Of HGW Gene Which Controls Flowing Time And Grain Weight In Rice

Rice is one of the world's most important food crops, and is considered as a model in Monocotyledons. Research on rice genes has both theoretical and practical significance.Heading date and grain weight are two determining agronomic traits of crop yield. Appropriate heading date can facilitate rice adapting to the environmental factors for higher yield. Gain weight is a critical factor of rice yield, while gain weight can be determined by grain size. Study on the mechanism of heading date and grain weight regulation is important for improving rice yield. To date, some molecular factors controlling both heading date and grain weight have been identified, but much has been unknown. In this study, we identify HGW by screening T-DNA insertion mutant labrary, which controls heading date and grain weigh in rice.The main study results were described as follows:1. We screen1100mutant lines in seedling stage. Unfortunately, we get no line with root mutant in which the phenotype was co-segregated with the T-DNA insertion. We get2lines, in which the reporter gene expression is specifically detected in root in seedling stage, and the expression is co-segregated with the T-DNA insertion.2. We isolate a hemizygous mutation, heading and grain weight (hgw), which delays heading and reduces grain weight in rice. We find that the heading date of this mutant is about20days later in natural field condition compared with WT control. The grain width of hgw is approximately75%of that in WT control. The final weight of1,000brown grains of this mutant is approximately78%of that of WT control.3. We get cross-section of hgw and WT hull, and we find that the overall circumference of hgw is approximately75%of that of WT control. Analysis of hgw mutant phenotypes indicates that the hemizygous hgw mutation decreases latitudinal cell number in the lemma and palea,19%and23%respectively. These results indicate that the main reason why the grain width of hgw is narrower is that the cell number is less.4. Southern blot analysis in TO hgw plant revealed a single T-DNA insertion in its genome.5. We isolate genomic fragments flanking the T-DNA insertion sites using thermal asymmetric interlaced PCR. We find that the T-DNA insertion is located in the first exon of LOC_Os06g06530. PCR test reveals that the mutant phenotypes co-segregate well with the T-DNA insertion.6. We perform genetic complementation experiments. No obvious heading date difference between the rescued individuals and WT controls was observed, as well as grain width and grain weight. We name the LOC_Os06g06530as HGW, which regulates heading date and grain weight in rice.7. Putative orthologs of HGW could be identified only in some plant, suggesting that the function of HGW and its orthologs is conserved during evolution of plants. The gene structure analysis reveals they have similar structure and there are5exons in most of them. More detailed protein sequence analysis reveals there is a UBA domain at the carboxy terminus of HGW and its orthologs. Whole-genome scale co-expression analysis in rice and Arabidopsis show that HGW and its Arabidopsis ortholog At5g53330are co-expressed with genes encoding various components of ubiquitin-dependent sorting and degradation pathways. These data suggest that HGW and their co-expressed genes have evolutionary conserved ubiquitination-related functions.8. To learn more about HGW at the subcellular level, we created HGW-YFP and HGW-RFP fusion proteins under the control of a constitutive CaMV35S promoter or a2.78kb native HGW promoter and studied the subcellular localization of HGW protein during transient expression in plant cells, including rice protoplast cells, tobacco leaf epidermal cells and onion epidermal cells. We find that HGW has a cytosolic and nuclear localization in rice cells.9. RT-PCR and Q-PCR analysis show that HGW is expressed in roots and leaves in both seedling and heading stages, as well as in sheath, stem and panicles at different development stages. GUS expression in the hgw mutant was detected in all tissues at the development stages, in line with the expression data described above.10. In hgw mutant, qRT-PCR analysis shows that the expression level of HGW in all tissues examined, including leaf, sheath, stem and panicle is significantly reduced compared to the WT control, suggesting that the expression level of HGW is essential for the regulation of heading date and gain width in rice.11. To gain insight into the molecular pathways regulated by HGW, we compare the transcript levels of OsGI, Hdl, Ehdl, and Hd3a in leaves in hgw and WT control by Q-PCR. These data imply that HGW differentially promotes the expression of Hdl, Hd3a and OsGI. 12. By analyzing the transcript levels of these genes that regulate grain size and weight including GIF1, GW2and GW5in hgw and the WT control with qRT-PCR, we found that expression of all these genes was reduced in hgw compared to the WT control.