S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Plant Science (식물생산과학부) Theses (Ph.D. / Sc.D._식물생산과학부)
Identification of SPLIT-HULL (SPH) and LARGE EMBRYO (LE) Genes in Rice (Oryza sativa L.)
벼의 개영(SPLIT_HULL) 유전자와 중거대배(LARGE EMBRYO) 유전자의 동정
- 농업생명과학대학 식물생산과학부
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 농업생명과학대학 식물생산과학부, 2018. 2. 고희종.
- Rice (Oryza sativa L.) is one of the most important cereal crop in the world. It is a primary food source of more than 90% of Asia and half of the worlds population. Since the edible part of rice is the grain (kernel), development of a grain from floral organs formation to grain filling is one of the most important topics in rice research and breeding. In this study, we characterized split-hull (sph) and large embryo (le) mutant and conducted map-based cloning to identify genes related to grain development and possibility on utilization of these genes to rice breeding.
Rice grain is divided into two parts, the hull and the kernel. Rice hull consisting of two bract-like structures, the lemma and the palea, protects the seeds from environment and determines kernel shape and size. The sph mutant showed hull splitting in interlocking part of lemma and palea during grain filling stage. Morphological and chemical analysis revealed that reduction in the width of the lemma and lignin content of the hull in the sph mutant might be the cause of hull-splitting. SPH was located in chromosome 4 and encoded type-2 13-lipoxygenase. SPH was expressed in leaf, stem, root, and spikelet, and interestingly, intensive expression was detected in the inner part of marginal region of lemma. The knock-out and knock-down transgenic plants showed split hull phenotype, and sph mutant contained significantly higher linoleic and linolenic acids (substrates of lipoxygenase) in spikelets than those of wild type. These results suggest that lipoxygenase (SPH) is involved in hull development and maintenance, and the genetic defect of lipoxygenase causes hull splitting phenotype. In dehulling efficiency test, the sph mutant showed high dehulling efficiency even by a weak tearing force in a dehulling machine, which suggests that hull splitting character might facilitate improving dehulling efficiency of fragile rice and breeding of easy-dehulling rice.
Rice embryo occupies a very small part of grain, but it contains various functional nutrients. Regulation of embryo size is an attractive breeding strategy to obtain dramatic improvement of rice nutritional quality. In the second chapter, three giant embryo mutants which were named according to the degree of embryo size enlargement as le, ge, and ges, from mild to severe, were morphologically and genetically characterized. They showed variation in embryo size, which was positively correlated with γ-aminobutyric acid (GABA) content. Allelism test and sequence analysis of GIANT EMBRYO (GE) locus revealed that ge and ges were allelic to GE which was previously reported as the gene controlling embryo size, whereas the enlarged embryo phenotype of large embryo (le) is controlled by a novel gene. Through map-based cloning, LE locus was delimited to the 51.8 kb region in chromosome 3, and one nucleotide deletion was detected in the 12th exon of OS03g0706900 encoding C3HC4-type RING finger protein. To investigate the suppression effect of LE gene, knock-down transgenic plants were generated and showed enlarged embryo seeds, suggesting that C3HC4-type RING finger protein is participated in the regulation of embryo morphology. LE was expressed in all organs examined such as leaf, stem, root, and predominantly in 20 DAP seed. Our findings provide the basis of new regulation mechanism determining embryo size and will facilitate the development of new giant embryo cultivars for improving nutritional quality of rice.