The Separation, Localization And Functional Analysis Of NADPH: Protochlorophyllide Oxidoreductase(POR) In Rice(Orvza Saliva L.)

A yellowish green leaf rice mutant, named as ygl3(yellow green leaf 3), was identified from the mutant library of Nippobare radiated by 60Coγ in this study. The phenotype of ygl3 was characterized, and the mutant gene was mapped and cloned. In addition, the tissue expression pattern, subcellular localization and functional relationship of mutant gene Os PORB and its homologous gene Os PORA were investigated1. Phenotypic analysis of ygl3The leaves of the ygl3 mutant are yellowish green at seedling stage and turning to yellow/white from the leaf-tip area during the late vegetative stages in the paddy field, and the fading degree is related to its degree of aging. The photosynthetic pigment content of the ygl3 mutant was measured at 10 days in seedling stage, the first and second leaf in heading period when the main spike came out completely, the first and second leaf in at 90 days in mature period. The total photosynthetic pigment content of the ygl3 mutant in seedling and mature period was significantly lower than the wild type, and the diminishing rate of chlorophyll b was higher than chlorophyll a.However, no difference was found for the total photosynthetic pigment content in the heading period between the ygl3 mutant and the wild type.2. The localization and cloning of mutant gene Os PORBIt was revealed that YGL3 gene was located on the region of 105.5 kb at the long arm of chromosome 10, by using the F2 population derived from the cross between ygl3 mutant as female parent and kasalath as male parent.The result of candidate gene sequencing analysis in this region indicated that a single base deficiency of a G which was occured at the 943 th of the fourth exon of Os PORB gene, this deletion produced a frameshift mutation and leaded to premature translational termination. For complementation of the ygl3 mutant, a complementary vector was constructured by Os PORA, Os PORB with the 35 S promoter. And the mutated phenotype of ygl3 could be complemented.3. The expression pattern of Os PORA and Os PORBWe thus examined the Os POR expression levels by the analysis of RT-PCR with the roots, stems and leaves at 10 days, the 1 to 5 leaf from bottom and spikes in heading stage of the wild type. It was found that expression of Os PORB wasconstitutive while the high level expression of Os PORA was occurred only in neonatal stems organizations and spikes. while it was gradually declined in the organization of the aging. the levels of Os POR expression were also examined in the roots, stems and leaves of the wild type and the ygl3 mutant. It was found that neither the expression of Os PORB nor the expression of Os PORA was changed due to the base deletion of Os PORB in the ygl3 mutant.4. The subcellular localization of Os PORA and Os PORBA fusion expression vector was constructured by Os PORA, Os PORB with EYFP(enhanced yellow fluorescent protein)driven by the 35 S promoter, transforming to the rice protoplasts instantaneously. It was evidenced that both Os PORA and Os PORB are located at chloroplast, they are chloroplast proteins.5. The functional of Os PORA and the relationship between Os PORAand Os PORBThe expression vector of Os PORA was constructed by RNAi technology and transformed to the wild type and the ygl3 mutant. The phenotype of Os PORA interference transgenic rice plant transformed from the wild type was the same as the wild type, having normal green leaves. The inhibition of Os PORA expression in the wild type did not affect the synthesis of chlorophyll. The phenotype of Os PORA interference transgenic rice plant transformed from the ygl3 mutant was yellowish green and nonviable. The synthesis of chlorophyll was completely blocked when the function of both Os PORA and Os PORB were missing.