| Maize is an important crop worldwide,which can be used for human food,animal feed,and biofuel production.It plays important role in people’s daily life.Photosynthesis mainly takes place in chloroplast and sugars synthesized in chloroplast directly or indirectly participate in every process of plant growth and development.The chloroplasts of higher plants include chlorophyll a and chlorophyll b.The synthesis of chlorophyll indirectly depends on trace elements such as iron,nickel and cobalt.The lack of these elements can affect the development of chloroplasts and further affect its functions.For example,plants may exhibit chlorosis when they are iron-deficient,and bleaching can also happen in some cases.Mutants with abnormal leaf color phenotypes are a type of mutants that show leaf color changes at different growth and development stages.Studies have found that leaf color mutants may be related to abnormal chlorophyll content or plant immunity.These mutants have gradually become good experimental materials for studying plant photosynthesis,photomorphogenesis,and exploring the color regulatory network of plant leaves.One zebra-necrosis mutant gn77 was obtained from the EMS mutant library.In this study,GN77 was characterized and cloned using map-based cloning and whole genome sequencing technology.The molecular function of GN77 was further studied and the research results were as following:1.Characterization of gn77:Compared with the wild-type material,the leaves of the gn77 mutant turned into light yellow-green about one week after germination,and formed irregular white spots.The mutant was stained with diaminobenzidine(DAB),and zebra-necrosis on the mutant leaves were stained brown,which indicated that a large amount of reactive oxygen species had accumulated in the mutant leaves.Real-time quantitative PCR(RT-qPCR)results show that the transcription levels of PR1 and PR5 in the gn77 mutant are higher than those of the wild-type,and the RNAseq analysis also found that the PR1 gene was lightly up-regulated.These results indicate that GN77 may affect the production of reactive oxygen species and potentially plant immune response.In addition,the gn77 plant is short and weak,indicating that the mutation of GN77 affected the normal growth and development process of plant height.Finally,the content of chlorophyll a and chlorophyll b in the leaves of the gn77 mutant material was significantly lower than that of the wild type.Transmission electron microscopy observations proved that the chloroplast structure of gn77 was destroyed,and some chloroplasts were degraded to a certain extent.Fewer thylakoids in the chloroplasts were detected or the thylakoids is only partial developed,indicating that the chloroplasts of gn77 were mutated.2.Map-based cloning of gn 7 7:statistical analysis of 160 F2 plants found that the ratio of wild-type phenotype and mutant phenotype plant number was in line with the genetic segregation ratio of 3:1(χ20.05<3.84),indicating that the phenotype is recessively controlled by a single gene.Then,the gene was crudely mapped to an interval of about 2.9 Mb between 9,429,841 and 12,350,606 on chromosome 1 using map-based cloning.As a result,in order to clone the candidate gene,the DNA pool of 39 gn77 mutants was used for whole-genome sequencing,and the SNP sites obtained by sequencing were used to develop dCAPS markers and Ture SNP markers;using a larger genetic population,the candidate genes were further mapped in the interval of about 780 Kb between 11,211,330 and 11,990,551.Bioinformatics analysis found that there were 10 genes in this interval.Through analysis of the EMS mutation sites in this region,a total of 20 potential EMS-induced mutation sites were found,but only the mutation sites located in the exon of GRMZM2G141636 can cause amino acid changes from proline to serine.The remaining mutation sites are located in introns or intergenic regions.Therefore,we preliminarily identified GRMZM2G141636 as a candidate gene for GN77.3.Allelic verification of GN77 gene:We obtained two allelic mutants of gn77 from the domestic maize EMS mutant library,named gn77-1 and gn77-2 respectively.The phenotype was investigated and found that gn77-1 homozygous mutant plants showed a clear zebra-necrosis phenotype,while gn77-2 did not have a zebra-necrosis phenotype.We crossed gn77 with gn77-1 and gn77-2 respectively for allelism test.Unfortunately,we only got the gn77-2/gn77 offspring,but failed to get the gn77-1/gn77 offspring.Phenotypic observation showed that the gn77-2/gn77 biallelic mutant showed a zebra-necrosis phenotype.We thus confirmed that GRMZM2G141636 is the candidate gene for gn77.4.GN77 encodes a chloroplast-localized nickel-cobalt transporter:Bioinformatics analysis of the protein sequence encoded by this gene revealed that GN77 contains 6 transmembrane domains and contains a conservative NiCoT sequence(HTLSGPDHL).Based on the protein database,the functional annotation of GN77 showed that it encodes a high-affinity nickel transporter.According to phylogenetic analysis,we found that the homologous protein of GN77 in Arabidopsis was a hypothetical high-affinity Ni2+/Co2+transporter.The results of subcellular localization analysis showed that the autofluorescence of chloroplasts was co-localized with the GN77-EGFP,which proved that GN77 was localized in chloroplasts.5.GN77 may affect photosynthesis of plants:In order to reveal the regulatory mechanisms in GN77,we performed transcriptome analysis.The results showed that there are 5,015 unique and significant differentially expressed genes between gn77 mutant and wild-type.Among them,2,876 genes were up-regulated and 2,139 genes were down-regulated.KEGG and GO(Gene Ontology)enrichment analysis showed that differentially expressed genes were significantly enriched in the photosynthesis pathway. |
