Identification,Gene Mapping And Cloning Of Leaf Yellow-to-Green Mutant In Maize

As a typical C₄ plant,corn produces 90%of its yield from photosynthesis of leaves.Photosynthesis refers to the process of green plants converting CO₂ and water into organic matter storage energy and releasing O₂ under visible light irradiation.It is an important way for plant carbon assimilation and biomass accumulation.Chloroplasts are important organelles for photosynthesis,and chlorophyll acts as an antenna pigment in cells to absorb and transmit light energy during photosynthesis.Discovering and identifying new genes related to plant chlorophyll synthesis and chloroplast development,cloning of photosynthesis-related leaf color mutant genes and their mechanisms of action will help us to understand plant chloroplast development,chlorophyll metabolism and photosynthesis.It has important theoretical and practical significance.The research material of this experiment was from the laboratory-created maize EMS mutant library,which was temporarily named vyl308（virescent-yellow leaf 308）according to the phenotype.The map gene was used to locate and clone the mutant gene,and the mutation was The phenotype and related physiological and biochemical indexes were analyzed,the cytological differences were observed,and the differentially expressed genes were mined by transcriptomics.The biological functions of the mutant genes were preliminarily studied.The results obtained are as follows:1.The phenotype of the vyl308 mutant is yellowing in the early stage of the leaf and turning green in the later stage.The leaf color gradually re-greens from the heart leaf area after the two leaves and one heart.The agronomic traits in the field showed that the mutants were lower than the wild-type RP125 in plant height,ear height,grain length,grain width and 100-grain weight,but the leaf area was slightly larger than wild-type RP125.The content of chlorophyll a and chlorophyll b in the leaves of yellowing green leaves were significantly lower than those of wild type RP125.The net photosynthetic rate,stomatal conductance and transpiration rate of mutants were significantly lower than wild type RP125.Mutant intercellular carbon dioxide was significantly higher than wild type RP125.The leaf color gradually recovered after the two leaves and one heart,and the pigment content showed a consistent recovery.The histological observation of the mutant leaves of yellowing and yellowing green leaves and the same parts of wild-type leaves in two-leaf one-hearted period showed that the development of mutant chloroplasts was blocked and the number of vesicles in thylakoids was reduced.2.The mutant vyl308 and the inbred lines B73 and Mo17 were crossed,and the F2 population phenotype was identified in the field.Genetic analysis showed that the yellowing phenotype of the vyl308 mutant was controlled by a pair of recessive genes.Using the F₂ generation clonal population constructed with B73 and Mo17background,the DNA of 20 yellowing plants and 20 normal plants was selected from the B73×vyl308 F₂ population by BSA strategy,and the cells were screened by the research group.More than 200 polymorphic SSR markers with polymorphism and uniform coverage of 10 pairs of chromosomes in the genotype were genotyped.The results showed that the SSR markers umc1072 and bnlg105a on the fifth chromosome were linked to the target gene,and the single genotype was combined.Analysis,preliminary determination of the target gene is located on chromosome 5 bin 5.08.At the same time,60 recessive individuals and 60 normal individuals in the F₂population of B73×vyl308 were mixed.Using BSRseq analysis strategy,it was found that there was a distinct peak on the chromosome 5 of maize,which proved the target gene.Located on chromosome 5,it coincides with the initial BSA results.Then,by searching the Maize GDB database for the published B73 reference sequence on chromosome 5 and the RP125 genome data obtained by the research group,a new SSR marker and Indel marker were developed,and the exchanged individual in the F₂population was used to finally locate the vyl308 gene in Indel.Between the MID23and the MID24,the physical distance between the two markers is 135 kb.3.In the corn database Gramene,the section was searched for 5 open reading frames,and each gene was sequence amplified and sequenced,and only mutations were found on ZmClpP6（Zm00001d018413）.There is a single base mutation in the fifth exon of the gene,which is mutated from"G"base to"A"base and mutated from glycine to glutamic acid.In order to verify the accuracy of the locus,the mutant single-strand DNA in the F₂ population of the mutant vyl308 was amplified and sequenced in an equal volume pool.The sequencing results were compared and verified,and all pool sequences were in this position.The point is still that the"G"base is mutated to the"A"base,and the sequencing peak map is a single peak at this site,which proves the accuracy of the site.The candidate gene for this mutant was initially shown to be ZmClpP6.4.By searching the maize genome database,the ZmClpP6 genome is 24,398 bp in length and contains 17 transcripts.The longest cDNA is 4846 bp in length.Of the17 transcripts,only 5 transcripts contain mutation sites,of which the longest transcript cDNA is 2071 bp in length,including 18 exons and 17 introns,encoding 292 amino acids,belonging to ClpP family protein.Looking up the gene function annotation of the gene,the gene is annotated as a molecular chaperone Hsp40/DnaJ family protein,and the encoded protein contains three domains of DNAJ,ClpP and Ribosomal L-9,wherein the mutation site is only in the ClpP domain.ClpP protein is an important protease that degrades damage proteins in chloroplasts.Phylogenetic tree analysis showed that the gene domain has higher homology with sorghum and rice than other organisms,which proves that the ClpP family gene is a very conserved gene,widely found in bacteria,cyanobacteria,plants and animals.The seedling development of plants is crucial.5.Real-time quantitative PCR was used to analyze the expression level of ZmClpP6 gene in different tissues.The results showed that the gene was expressed in most tissues of maize,but the expression level was the highest in leaves.The expression levels of some important genes related to photosynthesis（Sigma2A,Rpo2,II-1,photosystems Psb6,Psb29,Lhca1,Lhcb3 and Cab4）were analyzed.The expression levels of these genes in the mutants were lower than those in the mutants.The wild type indicates that the mutation of the ZmClpP6 gene affects the photosynthesis of vyl308.6.Select wild type and mutant samples with obvious phenotypes for transcriptome sequencing.After screening,252 differential genes were selected.GO and KEGG analysis were performed on the selected differential genes.It was found that most of the differential genes were enriched in photosynthesis-related pathways.In the above,it was proved that the ZmClpP6 gene is closely related to photosynthesis.7.Combining the phenotype of the mutant and related experimental results,it is speculated that the mutation is caused by the mutation of ClpP6 gene,which causes the function of the ClpP protein system to degrade the damage protein in the chloroplast,which leads to the hindrance of chloroplast development in the mutant.The reduction,which in turn causes the leaves to exhibit a yellowed phenotype,ultimately leads to a decrease in corn yield.As a key gene in the Clp protease system,the candidate gene ZmClpP6 has important significance and value for studying the stability mechanism of the Clp protease system.