Photosynthetic Properties And Gene Cloing Of Rice (Oryza Sativa) Albino Mutants Albi And Alblhc2

In higher plants, chloroplast is the place where photosynthesis takes place. Its development requires coordinated expression of both nuclear and plastid genes. Mutation of anyone of these genes would lead to the abnormal chloroplast development, resulting in changes of the photosynthtic properties. We managed to measure the photosynthetic property changes in rice albino muant albl and alblhc2, and to clone the mutated genes through a map-based method. By analyzing the gene functions, we tried to explain the relationship between gene mutation and the albino phynotype.1. Map-based gene cloning of an albino mutant and functional analysis of albl Through X-ray radiation, we got rice albino mutant albino lethality (albl) in the 9522(Oryza sativa spp. japonica) background. The homozygous rice mutant showed albino phenotype on its complete leaves and pale-green on incomplete leaves, which died within about 4 weeks after germination, while the heterozygous ones displayed normal phenotype. When the heterozygous plant was pollinated with wild type (WT) pollen, all F1 progeny were normal compared with WT descendants. F1 heterozygous seedlings were planted and self-polinated, all F2 progeny segregated with a ratio of 3:1. Genetic analysis indicated it's a single recessive nuclear gene that controls this phenotype.The mutant seedling was unable to grow photoautotrophically under a phytotron condition (30/24±1℃Day/Night, 50⁷0% R.H., and a Light/Dark period of 13/11 h at 100μmol photons·m-2·s-1). Chlorophyll fluorescence measurements showed a 30% decrease in maximum photochemical efficiency of PSII (Fv/Fm). Electron transport rate of PSII (ETRII) and PSI (ETRI) absorption indicated that the electron flow to photosystem II and photosystem I were inhibited. Blue-native PAGE indicated native photosystem complexes showed that few bands of photosystem protein-pigment complex in the mutant. Low-temperature (77K) fluorescence emission spectra also indicated that PSII and PSI were inhibited. We had a speculation that the malfunction of PSII and PSI were caused by degradation (or absence) of vital proteins, which was surported by western blot detection of many plastid genome ecoded proteins such as D1, D2, CP47, cytb559α, cytb6, PsaA, and PsaB.Through In-Del molecular markers, we cloned the albl gene by map-based method. In albl, it had a deletion of 5 nucleotides (CCATG) include the strat codon ATG of a PPR protein codon sequence. We took genomic DNA complementation with an 8,367 bp fragment (ALBL coding region plus a 1,971 bp upstream and a 3,816 bp downstream sequence) by Agrobacterium tumefaciens mediated transformation of the calli induced using the homogenous albl young panicles, the T2 seedlings showed WT phynotype. ALBL gene encodes a DYW-type PPR protein, which contains 11 PPR domains and an E domain, an E+ domain and a DYW domain. When fused the transit peptide to the N-terminal of YFP protein, ALBL was detected targeting to the chloroplast.By ALBL antibody, we pulled down 4 candidate chloroplastic proteins (J090072P03, Os03g0214900, OsJ₁9331, and Q09HW4) which interact with ALBL in vivo. We checked the relationship between ALBL and each of the candidates by yeast-two-hybrid LexA system, and finally found Os03g0214900 (SRI), which has a C-teminal SRI (Set2 Rpb1 Interacting) domain, was the real protein that mediates ALBL to be functional. Further research on the interact region(s) and the interaction mechanism is being taken. 2. The photosynthetic property and molecular cloning of rice alblhc2 mutantAn albino rice mutant was isolated using isotope mutagenesis strategy in the 9522 (Oryza sativa spp. japonica). Genetics analysis indicates that it's a single recessive nuclear gene, named alblhc2 that controls this phenotype. In mutant 2-week old seedlings, nearly all the trimer of Light-Harvesting Complex II (LHCII) degraded to monomer, many other protein complexes are also invisible when the thylakoid total proteins were separated by blue-native PAGE. Chlorophyll fluorescence measured showed the dark level fluorescence (Fo) increased remarkably and maximum photochemical efficiency of PSII (Fv/Fm) decreased by about 60% in mutant. Electron transport rate of PSII (ETRII) absorption indicate that the electron flow to PS II is inhibited. The malfunction of PSII was also supported by the severe decrease in many PSII proteins analyzed by western-blot. Through In-Del molecular markers, alblhc2 was mapped in a region of about 1,535 kb on chromosome 4 between marker Os408 and Os40719 by map-based cloning. The clone and functional analysis of this gene will be further taken.