Screening For Arabidopsis Thaliana Leaf Color Mutants,Cloning And Functional Characteration Of One Related Gene

Chloroplasts are the sites where photosynthesis takes place and many organics synthesize,providing essential materials and energy for human survival and development.So studies on chloroplast development are of great significance.Chloroplast development and leaf color are closely related.Deficiency in chloroplast development can often lead to content abnormalities of photosynthetic pigments and further results in leaf color variation.The aim of this study is to clone mutated genes which may relate to chloroplast development via screening for leaf color mutants from EMS mutagenized Arabidopsis populations and further explore their functions and molecular mechanisms in chloroplast development.Main results of this study include the following parts:(1)16 leaf color mutants were obtained,including types like pale-green,yellow-green,netted-variegation,patched-variegation,dark-green,stay-green etc.and backcrossed with wild type Arabidopsis to analyze their inheritance patterns and purify their genetic backgrounds;(2)Positional cloning was conducted to find the mutant gene of a yellow-green leaf color mutant numbered F05-44.A point mutation was found in the seventh intron of the gene ERA-1.This mutation leads to the sequence of the first 30 nucleotides of the seventh intron,which contains a stop codon,remaining in the mRNA and results in premature translational termination.Genetic complementation experiments confirmed that the phenotype of F05-44 was caused by the mutation in ERA-1;ERA-1 protein tagged with eGFP was transiently expressed in mesophyll protoplasts of Arabidopsis,and the results confirmed that ERA-1 is located in chloroplast;ERA-1 coding sequence,except the chloroplast targeting peptide coding part,was cloned into prokaryotic expression vector pET28 a,and then transformed into E.coli BL21(DE3).Recombinant protein Hisx6-ERA-1 was obtained to immunize rabbits as antigen to harvest ERA-1 antiserum.Polyclonal antibodies were purified by their affinity with Hisx6-ERA-1.Western blotting results show that the antibodies prepared in this experiment(diluted to 1/1000)can detect ERA-1 specifically in total leaf protein preparation of wide type Arabidopsis,and thus can be applied into future studies.In conclusion,the leaf color mutants obtained from this study can be useful plant materials for future studies on chloroplast development.Experiments on subcellular localization and polyclonal antibodies preparation of ERA-1 provide clues and advantages for further studies on the function and molecular mechanism of ERA-1.