| This thesis includes two parts:the first is "map-based cloning and functional analysis of YGL2gene for yellow-green leaf in rice", and the other part is "fine mapping of a QTL qPGWC-7for percentage of grains with white chalkiness and preliminary characterization of the candidate gene in rice".Part one:Heme Oxygenase (HO) in higher plants is a rate-limiting enzyme for catabolism of heme, it can catalyze the degradation of heme to synthesize phytochrome precursor and its roles conferring the photoperiodic control of flowering in rice have been revealed. However, its involvement in regulating rice chlorophyll (Chi) synthesis is not fully explored. In this study, we isolated a rice mutant named yellow-green leaf2(ygl2) from a Co-irradiated population. Normal grown ygl2showed yellow-green leaves in seedling stage and was most obvious in three-leaf stage, from tiller initiating to heading stage, yellowish leaves gradually turned green and approached almost as normal as wild type did. Pigments contents at different developmental stages were examined and the results showed that, at three-leaf stage, the ygl2mutant had a significant reduction of Chi and Car levels but apparent increase of Chi a/b ratio compared to the wild type but these difference were decreased along with the plant grown. We compared the ultrastructures of chloroplasts between the ygl2mutant and wild type plants at three-leaf stage using transmission electron microscopy. The number of thylakoid lamellar in chloroplasts of the ygl2was significantly less than that of wild type, whereas the structure of the thylakoid lamellar seemed normal and similar to that of wild type. For map-based cloning of ygl2gene, a mapping population of F2was constructed by a cross between the ygl2mutant and Nipponbare, genetic analysis indicated that the chlorosis phenotype in the ygl2mutant was controlled by a recessive nuclear gene. The ygl2locus was finally narrowed to a region of66.8-kb between two InDel markers, In44and In39on the long arm of chromosome6. Sequencing analysis showed that a fragment of7-kb was inserted into the first exon of one gene in this region which encode Heme Oxygenase, resulting in amino acid polymorphisms and expression level declined. Genetic complementation and RNAi all indicated that the abnormal phenotype of ygl2mutant resulted from the mutation of YGL2gene. Phylogenetic tree analysis showed that YGL2belong to the HO1family, and the mutation of YGL2made little changes to the secondary and3D structures of the protein, but had no effect on the localization of chloroplast through subcellular localization of rice protoplast. qRT-PCR and GUS histochemical staining indicated that YGL2were constitutively expressed in various tissues and particularly the highest in leaf. The expression level at seedling stage was much higher than that of tillering stage and that it was affected obviously after treatment with two different temperatures, meanwhile, the expressions of genes associated with Chl biosynthesis and photosynthesis were also changed. The results indicated YGL2played an important role in the regulation network of Chl biosynthesis and photosynthesis. The cloning of YGL2established the foundation for illuminating the important roles of HO in regulating Chl biosynthesis and photosynthesis in higher plants.Part two:Chalkiness of rice grain has profound influence on qualities of rice, it is a complicated quantitative trait and controlled by many quantitative trait locus (QTLs). Fine mapping and cloning of QTLs for chalkiness not only helps us in illuminating the molecular mechanism of chalkiness, but also improving breeding efficiency using marker-assisted selection (MAS). In this study, C-51was a chromosome segment substitution lines (CSSL) created using9311as the recurrent and PA64s as the donor, the percentage of grains with white chalkiness (PGWC) of C-51was significantly higher than that of9311in different environment. Scanning electron microscope (SEM) analysis showed the C-51starch granules of opaque part were round or oval, loosely packed and sizes, and very different from angular, densely packed, polyhedral starch granules of9311transparent part, but there were no significant differences in fresh weight of grain and dry weight of brown rice between C-51and9311in different periods after flowering. We verified the results previously and conducted further fine mapping. The QTL qPGWC-7was finally narrowed to a region of17.1-kb between two InDel markers, InDel18and InDel3on chromosome6. This region contains three ORFs, the expression of ORF1in C-51was much higher than that of9311during grain filling period, especially the highest 18days after flowering. In addition, there was a amino acid polymorphism in the conserved domain of ORF1protein between C-51and9311, so ORF1was used as the candidate gene for further functional analysis. The enzyme activity of qPGWC-7was no difference between expressed proteins of C-51and9311in vitro, so the difference for chalkiness may caused by the difference of expression levels. GUS histochemical staining indicated that qPGWC-7was constitutively expressed in vascular tissues of various organs and particularly the highest in young panicle. On the other hand, the expression level was induced by low temperature. qPGWC-7was located on Golgi through subcellular localization of rice and Arabidopsis protoplast. These results above provided a good basis for further study of qPGWC-7function. |
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