Map-based Isolation And Characterization Of Key Genes Involved In The Glutelin Biosynthesis Pathway In Rice

Rice (Oryza sativa L.) seeds accumulate large amounts of storage proteins (seed storage proteins, SSP) in endosperm during seed development. Glutelins account for about 80% of the total proteins and deposite in protein body II (PBII). Any change in glutelin content or composition will alter rice quality. Although most of the glutelin genes or cDNAs had been cloned, the expression and regulation of glutelin genes and the cellular processes underlying glutelin biosynthesis, transport and deposition remain to be understood. The development and utilization of mutants combining the strategy of map-based cloning is one efficient approach in resolving above problems. To obtain mutants, over 9,000 lines including landraces, T-DNA insertion lines and radiation induced mutation lines were subjected to SDS-PAGE analysis. Totally we obtained 17 mutants, of which 3 ones have been studied extensively. To the low glutelin content mutant, W3660, we fine-mapped the mutated gene, and applied the flanking markers in marker-assisted selection. To the 57H mutant, W379, we isolated the mutated gene through map-based cloning strategy and elucidated the molecular mechamism underlying glutelin precursors accumulation in W379. To another 57H mutant, Q4041, we also map-based isolated the mutated gene, and a microarray experiment was done to search for the reason why the 57kD precursor was partly accumulated and to explore the affected pathway(s) caused by the mutation. The results are as followings: 1. Screening of Seed Storage Protein mutantsAfter screening over 9000 lines, 17 SSP mutants were obtained with a mutation fequency of only 0.189%. These mutants can be simply classified into 3 types: a, the LGC mutant which is characterized by low content of glutelins and high content of prolamins and globulin. It can be used as not only the donor parent in developing a rice cultivar suitable for patients with diabetes and kidney disease, but fairly good materials for isolation genes responsible for the expression and regulation of glutelin genes as well; b, the 57H mutant which is characterized by high amount of 57kD polypeptides and low amounts of mature glutelin subunits. The seven 57H mutants are excellent materials for isolating key genes involved in the regulation of transport and deposition of glutelins; c, the globulin deletion (gd) mutant which is characterized by the missing of globulin polypeptide. This may be caused by the mutation of globulin itself or gene(s) regulating the globulin biosynthesis. Moreover, pyramiding genes of Lgc and gd will develop a cultivar with even lower amount of soluble proteins.2. Fine mapping and marker-assisted selection of a low glutelin content gene in W3660Rice with low glutelin content is suitable as functional food for patients affected with diabetes and kidney failure. The fine mapping of the gene(s) responsible for low glutelin content will provide information regarding the distribution of glutelin related genes in rice genome and will generate markers for the selection of low glutelin rice varieties. W3660 is a novel mutant characterized by low glutelin content. F₂ and F₃ populations were derived from a cross between W3660 and Jingrennuo to map the gene(s) responsible for the low glutelin content. SDS-PAGE analysis of the total endosperm protein showed that the low glutelin content trait was controlled by a single dominant nuclear gene. Genetic mapping, using SSRs, located this gene to chromosome 2, in the region between SSR2-001/SSR2-004 and RM1358. The distances of the two markers to the target gene were 1.1 cM and 3.8 cM respectively. Although the chromosome position of this gene was similar to LGC-1, they are different genes. SSR profiles of seven japonica varieties were compared with that of W3660 for loci in the relevant genetic region. The markers SSR2-004 and RM1358 were used for marker-assisted selection. The selection efficiencies of SSR2-004 and RM1358 were 96.8% and 92.7% respectively, and thus these markers will be suitable for the selection of low glutelin content rice.3. Isolation and characterization of a vacuolar processing enzyme responsible for the maturation of glutelins in riceRice accumulates prolamins and glutelins as storage protein. The latter storage proteins are synthesized on rough endoplasmic reticulum (rER) as 57kD precursors and are sorted into protein storage vacuoles, where they are processed into acidic and basic subunits forms. We report here a rice 57H mutant, W379, which abnormally accumulates glutelin precursors. Genetic analysis revealed that the phenotype in W379 was controlled by a single recessive nuclear gene. Through map-based cloning, a homolog of the arabidopsisβVpe was isolated, which was tentatively nominated osVpe1. The coding sequence of osVpe1 has 1494 base pairs, and encodes a protein of 494 amino acids. Sequence analysis showed that only one nucleotide was different between W379 and Nipponbare, resulting in a change of Cys269 to Gly. The expression pattern and expression level of osVpe1 in developing endosperm were very similar in the two cultivars. Subcellular localization indicated the deformed protein was also sorted into vacuoles as WT protein did. But the enzyme activity was almost lost in W379. Western blotting revealed that osVpe1 was cleaved by mistake, resulting in a smaller size of mature protein. Our findings suggest that osVpe1 plays a crucial role in the maturation of rice glutelins, and the Cys269 residue is a key residue in maintaining its' Asn-specific cleavage activity.4. Isolation and Characterization a small GTP binding protein, osRab5a, involved in the transport of storage proteins in riceA new 57H mutant named Q4041 was obtained from radiation induced mutation lines, Q4041 abnormally accumulated glutelin precursors, but its' amount was less than W379. The seed of Q4041 was smaller and had opaque endosperm. This indicated a defect in starch biosynthesis. SEM analysis of the mature endosperm showed that the opaque endosperm comprised round and loosely packed, compound starch granules. Genetic analysis showed the mutant trait in Q4041 was controlled by a single recessive nuclear gene, the 57H trait and opaque trait were controlled by the same gene. An F₂ population derived from the cross between Q4041 and Reyan2 was adopted to map the gene responsible for the 57H trait. Using PCR-based molecular markers, we mapped the gene on Chromosome 12, in the region between 12-170 and 12-191, with a physical distance of 27kb. In this region, three genes were predicted, including osRab5a, ubiquitin E3 ligase gene and a retrotransponson gene. Sequence analysis showed no differece in E3 ligase gene between WT and mutant. Semiquantitative RT-PCR analysis showed that the expression of osRab5a in Q4041 was much lower than in WT. Further we sequeced the genomic DNA and cDNA of osRab5a in Q4041 and found a 13bp deletion in the third exon. Thus we obtained the gene responsible for the 57H trait in Q4041. To explore the affected pathway(s) caused by this mutation, a microarray experiment was done, and found that totally only one gene was downregulated and over 1000 genes was upredulated in Q4041 compared with WT(Foldchange>2 or foldchange<0.5, and P<0.05), including osRab5a itself, sugar metabolism and starch biosynthesis related genes, Calcium binding proteins, peroxidase gene family, Heat shock proteins, and Eukaryotic transcription initiation factor, etc. This provides a basis for the characterization of osRab5a.