| RNA editing is a post transcriptional modification process for DNA encoding genetic information,which could cause the nucleotide insertion,deletion or replacement during the maturation of RNA.Through RNA editing,a gene produces a variety of encoded proteins to keep their genetic stability,which help plants to adapt to environment changes by enriching the genetic information.RNA editing exists extensively in organisms,and RNA editing of higher plants mainly occurs in mitochondria or chloroplasts,which is an important event of the evolution of plant genetic mechanism.There are some reports on PPR protein regulating RNA editing in chloroplast and mitochondria of Arabidopsis,rice and maize.However,the study of PPR protein in soybean has been not reported yet.In this thesis,we report identification of Glycine max pale green 1?Gmpg1?,and cloning of GmPG1 gene,which encodes a PPR protein participating in chloroplast RNA editing.The mechanism of GmPG1 to regulate chloroplast RNA editing is also investigated,which is the firstly reported work of PPR protein in RNA editing of soybean.The major results are described as following:1.Isolation and genetic analysis of soybean Gmpg1 mutantBy screening a gamma mutagenesis population of soybean cultivar,Hedou12,we identified a pale green leaf mutant,named as Gmpg1?Glycine max pale green?.The contents of total chlorophyll,chlorophyll a and b of the Gmpg1 mutant were 1.08mg/g.FW,0.69 mg/g.FW and 0.38 mg/g.FW respectively,which were only 59.6%,57.3%and 64.3%of wild type,Hedou12.The photosynthetic rate of Gmpg1 was 3.41?mol CO2m-2s-1,which was decreased by 70.6%compared with Hedou12.The PSII maximum quantum yield of Gmpg1 was only 45%of that in the Hedou12.The number of thylakoid membranes decreased in Gmpg1.Genetic analysis indicated that the phenotype of pale green leaf,abnormal chloroplast and photosynthesis decreased was controlled by a recessive single locus.2.Mapping and Cloning of GmPG1 geneThe Gmpg1 mutants were crossed to Williams82 to generate a F2 segregation population which contains 270 individuals.Using the mapping-based cloning method,the mutated locus of GmPG1 was initially located on chromosome 05 in the region of31.1-38.0M;after fine mapping,this locus was located in the region of 32.44-32.59M.In this region,there are 15 predicted genes;whole genome sequencing revealed that an A deletion was detected at 1949bp from the start codon in the Glyma.05g132700.1 gene,while none mutation was detected in the remaining 14 genes in the candidate region.This deletion would cause a frame shift and premature terminate code,which lead to680 amino acids instead of 742 amino acid.We constructed the overexpression plasmid of GmPG1 and transformed into the Gmpg1 mutants,transgenic T2 progeny transgenic plants restore the green leaves as wild-type plants,indicating that Glyma.05g132700.1 was a gene that responsible for Gmpg1 phenotype.3.Protein function and subcellular localization of GmPG1The predicted GmPG1 protein is an atypical PPR-DYW protein which containing fifteen PPR or PPR-like?P,L and S?domains and a C-terminal DYW domain.A deletion in 1949bp of Gmpg1 gene lead to DYW domain deletion.We constructed 35S:GFP-GmPG1P and transient expressed in Arabidopsis protoplasts,GFP-GmPG1P co-localized with the auto-fluorescent signals of chlorophyll,suggesting that GmPG1protein was located to the chloroplasts in soybean.4?RNA editing of soybean during different leaf development stagesAnalysis of the RNA-seq data indicated that 42 chloroplast genes were edited,40 of the 42 soybean plastid RNA editing sites are located in the coding region of 18 different genes,and editing of each of the 37 sites results in an amino acid change except for ndhB-273,ndhC-4 and rps7-300.The 37 RNA editing sites results in five types of amino acid change including serine-to-leucine amino acid,proline-to-leucine amino acid,serine-to-phenylalanine amino acid,serine-to-methionine amino acid and histidine-to-tyrosine amino acid.The other two editing sites,rps12-540 and rps16-885are located in intron,respectively.RNA editing efficiencies are different in different editing sites for two developmental stages such as ndhB-586,ndhB-737 and ndhD-2which RNA-editing efficiency up by more than 50%in the second developmental stage.Most of editing sites are edited in two developmental stages except for ndhB-273,rpo C2-2735,rps7-300,ycf2-3671 which are only edited in the second developmental stage.The results indicate that RNA editing exist extensively in chloroplast of soybean and RNA editing efficiency have variations in different development stages.5?GmPG1 regulated chloroplast RNA editing of soybeanThere chloroplast genes,rps16-212,ndhF-290,ndhB-1255,showed significant differences of RNA editing efficiencies between wild type and Gmpg1 by high resolution melting curves?HRM?examination of above chloroplast RNA editing sites.The RNA editing deficiencies of the Gmpg1 were restored in the Gmpg1 complemented transgenic lines.rps16 encodes small ribosomal subunits and is an essential plastid gene that important for translation process.The cytidine?C?-to-uridine?U?editing of chloroplast rps16 was found at 212bp of CDS,produced a serine-to-leucine amino acid changed.In the leaf early development stage,the RNA editing efficiency of Gmpg1 was 40%which reduced 42.8%compared with HD12,and in the late leaf development stage,the RNA editing efficiency of Gmpg1 was 0%which decreased 100%compared with HD12.NdhF encodes the subunit of the chloroplast NAD?P?H dehydrogenase?NDH?complex.The cytidine?C?-to-uridine?U?editing of chloroplast ndhF was occurred at290bp of CDS,resulting of histidine-to-tyrosine amino acid.In the early development stage,the RNA editing efficiency have no different significant between Gmpg1 and HD12,in the late leaf development stage,the RNA editing efficiency of Gmpg1 reduced22.2%compared with HD12.NdhB encodes the subunit of the chloroplast NAD?P?H dehydrogenase?NDH?complex.the cytidine?C?-to-uridine?U?editing of chloroplast ndhB was occurred at1255bp of CDS,resulting of serine-to-leucine amino acid.In the two development stages,compared with HD12,in the early development stage,the RNA editing efficiency of Gmpg1 have no different significant,however,in the late development stage,the RNA editing efficiency of Gmpg1 reduced 44.4%.These results indicated that GmPG1 influenced chloroplast development and photosynthesis by regulation of RNA editing of rps16-212,ndhF-290,ndhB-1255... |
PDF Full Text Request