The Gene Identification Of An Arabidopsis Thaliana Delayed-green Leaf Mutant And Its Preliminary Fuction Study

Chloroplast is not only the specific organelle of plant,but also the site of some important metabolism pathway,such as photosynthesis,photorespiration and nitrogen assimilation.Therefor to study the development of chloroplast is very important to understand the plant development and production.In this study,a leaf-delayed green mutant was screened from EMS mutagenized Arabidopsis thaliana Col-0 M₂ pools.The new leaves of this mutant are yellowish,and the yellowish leaves can turn to green along with development.The development of chloroplast was studied by using this mutant.1.The identification of mutated geneThe delayed-leaf mutant?Col-0 background?was crossed with Ler wild type to generate the mapping population.By using F2 plants with phenotype,the mutated gene was roughly mapped between 3.83M and 8.00M on chromosome 1,and the mutated gene was further mapped between 5.3M and 5.83M on chromosome 1 by fine mapping.Combining the genome resequencing results,there is a SNP of G to A at the 1931th base of At1g15510gene(Gly⁶⁴⁴intoAsp⁶⁴⁴)identifiedinthisregion.Forfurther confirmation,theAt1g15510 gene sequence was downloaded from the Tair web site?www.ababidopsis.org/?.The 1936 bp promoter sequence and the full length of the gene were ligated to construct complementation vector.The phenotype of positive transgenic lines is rescued by At1g15510 gene.The phenotype of this mutant is caused by the SNP mutation of At1g15510.Thereforthis mutant is namedecb2-3.2.AtECB2 gene is a member of the PPR familyAccordingtotheArabidopsisPPRprotein data?http://www.plantenergy.uwa.edu.au/applications/Ppr/ppr.php?,the AtECB2 protein belongs to the DYW subclass of the PLS subfamily of the PPR family of proteins,with 17PPR motifs.Compared withthe ECB2 homologous protein sequences of different species,it was found that the mutated Gly in ecb2-3is a non-conserved amino acid.3.The chloroplast development of ecb2-3 mutant is affectedThe chlorophyll content and the maximum photochemical efficiency of the mutantswere measured.The results showed that the chlorophyll content and maximum photochemical efficiency of the green leaves of the mutant are not significantly different from that of the wild type,however there are significant difference compared with the yellowish leaves.The ultrastructure of chloroplast of mutant was also observed by transmission electron microscopy?TEM?.The results showed that the chloroplasts of ecb2-3 cotyledons were less than the wild type chloroplasts at 7 days after germination.In the ecb2-3 mutant after 21 days of germination,the chloroplasts of yellowish leaves lack thylakoid membrane and starch granules,and the chloroplasts of green leaves contain thylakoid membranes and starch granules similar to those of wild type.In the complementary plants,the chloroplast has a similar ultrastructure as the wild type plant.4.RNA editing efficiency of accD and ndhFin ecb2-3 mutant decreasedIt has been reported that AtECB2 is involved in RNA editing of Arabidopsis thaliana chloroplast gene accD?C794 and C1568?and ndhF?C290?.We extracted the Col-0wild-type,ecb2-3 mutant RNA and reverse-transcribed into cDNA to amplify the editing sites of accD and ndhF with the appropriate primers designed for cDNA sequencing,and dCAP primers at the editing sites were designed to analyze their editing effiency.The results showed that the editing efficiency of the mutants at these three sites is lower than that of wild type.These results indicate that AtECB2 is necessary for Arabidopsis thaliana chloroplast accD gene and ndhF gene RNA editing and early development.Owe to the weak allele of ecb2-3 mutant and its genetic stability,this mutant will be useful in further studies of Arabidopsis thalianachloroplast RNA editing and chloroplast development.