Map-based Cloning And Functional Analysis Of True-leaves-etiolation Mutant Cfl1in Arabidopsis Thaliana

In higher plants, chloroplast is the place where photosynthesis takes place. The chloroplast genomesof higher plants typically encode only about100genes, and the majority of the chloroplast proteins thathave functions in the chloroplast are encoded by nuclear genes, translated in the cytosol and subsequentlyimported into the chloroplast. Thus, the development of functional chloroplasts is dependent on thecoordinated expression of nuclear and chloroplast genes. Studies into the processes of biogenesis anddevelopment of chloroplasts have shown how complex the chloroplast signal net is during plantdevelopment. However, we know little about its development molecular mechanism. Thus, it is necessaryto understand the regulation and mechanism of chloroplast development.In this research, Arabidopsis thaliana ecotype Columbia-0was used and a mutant library wasestablished by treating Arabidopsis Columbia-0seeds with chemical mutagenesis agent sulfonic acid ethylester (EMS). We used chlorophyll fluorescence imaging to screen and separate a lower chlorophyllfluorescence value mutant when compared with wild-type plant, we termed this mutant cfl1(chlorophyllfluorescence lower1). Further study showed that the true leaves of cfl1were deformed, its true leaves werewhite variegated, the leaf margin of the mutant were more serrated and asymmetrical. cfl1grew moreslowly than wild type plants, the production of the mutant and plant height were lower than wild type.Physiological indexes measured results showed that chlorophyll content in white variegated true leavesdecreased significantly and the conductivity increased significantly when compared with wild type. Biopsyresults showed that chloroplasts structure in cfl1white variegated leaves was incomplete that exhibited nograna and stroma thylakoid. Genetic analysis showed that variegation phenotype in cfl1was due to a singlerecessive gene mutation. We crossed cfl1(♀) and Landsberg erecta (♂) to construct a collection of F2which were used to map the mutant gene. The mutant gene finally was resided on BAC (Bacteria ArtificialChromosome) MWD9through map-based cloning. Then we got the mutant gene CFL1by gene sequencing,found one of its bases G changed into A at its1145site by blasting with the sequence provided in TheArabidopsis Information Resource. And the matched amino acid changed into Ser (AGT) from Gly (GGT).Genome structure of CFL1consists of three extrons and two introns, encoding an unknown protein. Tofurther examine the function of CFL1in Arabidopsis development, we constructed all kinds of vectors such as complementation vector, over expressing vector, and transient expression vector etc. We examined theexpression profiles of chloroplast photosynthetic marker genes in different development stages by RT-PCR,and the result indicated that the expression of chloroplast photosynthetic genes were down-regulated.Taken together, these data indicated that CFL1was a new player in chloroplast development, and thegain of this mutant provide us an ideal material to conduct further research on the function of this gene andthe development mechanism of chloroplast.