| Most of rice yield comes from leaf photosynthesis,which depends on the normal development of chloroplasts.Chloroplasts are semi-autonomous organelles that are generally considered to be endosymbiosis of photosynthetic bacteria.During endosymbiosis,most of the prokaryotic genome is lost or transferred to the nuclear genome,and most chloroplast proteins are encoded by nuclear genes.Only transported to specific functional sites chloroplast proteins can perform their functions.Chloroplast proteins encoded by nuclear genes are first transported to chloroplasts by complexes on the inner and outer membranes of chloroplasts,and then transported to thylakoids by different transport pathways.Therefore,the study of chloroplast development and chloroplast protein transport mechanism is helpful in understanding the regulation mechanism of rice photosynthesis,and lay a foundation for further cultivation of high-yield rice.In this study,a leaf yellowing mutant yll1(yellow leaf and lethal 1)was selected and the target gene OsALB3(ALBINO3)was cloned.The function of OsALB3 is preliminarily studied,and the main conclusions are as follows:1.The yll1 mutant was isolated from the offspring of the japonica rice(Oryza sativa)variety,Nipponbare treated with EMS(Ethane Methyl Sulfonate).At the seedling stage,the leaves of yll1 were light yellow and yll1 died 24 days after germinating.The content of chlorophyll a and b in the yll1 was significantly lower than that of the wild type.The chloroplast structure of yll1 was abnormal and the number of thylakoid membranes was significantly reduced.2.Genetic analysis showed that the leaf yellowing character of yll1 was controlled by a recessive nuclear gene.DNA of the two pools derived respectively from the 30 wild type and yll1 plants in the same population were subjected to high-throughput sequencing,valid sample data were screened by MutMap+ technology.According to the specific rules of SNP/Index-index=1 in mutant pool and SNP/index-index<0.5 in wild type pool.Finally,the only candidate gene LOC_Os01g05800 was screened.3.In mutant yll1,a single base substitution,C to T,resulted in the mutation of the 140th amino acid from alanine(Ala)to valine(Val).According to the rice genome annotation LOC_Os01g05800,it is similar to the inner membrane protein encoding gene ALBINO3 in Arabidopsis thaliana.Therefore,we named this gene OsALB3.OsALB3 contains 12 exons with a 60Kd inner membrane protein domain in the central part.BLAST found OsALB3 homologues in plants,animals,fungus and bacterium.Multiple sequence alignment analysis showed that the OsALB3 homologous proteins in different species were highly conserved in the 60Kd intimal protein domain.OsALB3 gene was edited by CRISPR/Cas9 system,and the knockout mutant was obtained.Its phenotype was consistent with that of the mutant yll1,further proving that the OsALB3(LOC_Os01g05800)was the target gene.4.QRT-PCR results showed that OsALB3 gene was expressed in all tissues of rice,and its expression level was higher in leaves.Subcellular localization showed that OsALB3 protein was localized in chloroplasts.5.In Arabidopsis Thaliana,it has been shown that ALB3 is mainly responsible for integrating light-harvesting chlorophyll binding proteins into the thylakoid membrane in the chloroplast signal recognition particle pathway,thus further participating in photosynthesis.Therefore,the expression level of related pigment proteins in yll1 was analyzed by Western-Blot experiment.The contents of light-harvesting pigment proteins Lhca1,Lhca2,Lhca3,and Lhca4 in photosystem Ⅰ(PS Ⅰ)and photosystem Ⅱ(PS Ⅱ)were significantly reduced in mutant yll1.Therefore,OsA LB3 plays an important role in the accumulation of light harvesting chlorophyll binding protein,which provides a reference for the further study of transport mechanism of chloroplast protein and lays a foundation for the regulation of photosynthesis in rice. |
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