| 1.Map-based cloning of yellow-green leaf mutant 520ys in riceThe chloroplast is the basic unit of the structure and function of plant photosynthesis,and chlorophyll is the important pigment involved in photosynthesis in plant chloroplasts.So the transform of chlorophyll content is directly related to plant photosynthesis.The mechanism of chloroplast development and the synthesis pathway of photosynthetic pigments can be understood through the study of leaf color mutants,and then in-depth study of rice photosynthesis mechanism,which can provide theoretical basis for plant high light efficiency and improve the yield of high light efficiency rice varieties way.In our study,a rice yellow-green leaf mutant 520ys was obtained by mutagenization of EMS.The mutation mechanism of 520ys was studied by genetic analysis,gene mapping,candidate gene screening and functional complementation.The results were as follows:(1)The phenotype observation and agronomic traits analysis of mutant:520ys displayed a yellow-green phenotype of the entire growth period and growth retarded;the heading period is delayed.Agronomic traits survey results show that:the effective panicle,seed setting rate and thousand grain weight were very significantly decreased by 20.0%,11.6%and 5.4%respectively,the plant height decreased significantly by 3.6%.These indicating that 520ys mutant gene had a great influence on plant growth.(2)Determination of photosynthetic pigment content in mutant:compared to its wild type,the 520ys photosynthetic pigment content was very significantly lower at seedling stage and heading stage,wherein seedling chlorophyll a,chlorophyll b,carotenoids and total chlorophyll content decreased by 52.5%,79.5%,29.3%and 58.0%,the chlorophyll a/b ratio increased by 124.3%;and heading stage of chlorophyll a,chlorophyll b,carotenoids and total chlorophyll content decreased by 59.5%,82.9%,59.2%and 63.7%,chlorophyll alb ratio increased by 143.1%.These description 520ys mutant photosynthetic pigment synthesis are blocked.(3)Ultrastructural observation of chloroplast:the transmission electron microscopy of leaves at 4-week-old seedling showed that:520ys chloroplast thylakoid development disorder,no significant lamellar stacks,and wealthy in vesicular structure,significantly increasing the number of osmiophiilic granule.Indicating that the 520ys mutant chloroplast development was inhibited.(4)Genetic analysis and gene mapping of mutant:520ys mutant traits was controlled by a pair of recessive nuclear genes,and the genetic loci were located on the long arm of chromosome 7 and within the range of 167.6 kb between the InDel marker X3 and RM21725,the genetic distances were 0.60 cM and 0.42 cM.(5)Screening,validation and bioinformatics analysis of candidate genes:a total of 21 genes in the range of 167.6 kb were sequenced and analyzed.It was found that the substitution of a base of unknown protein gene in the region was changed from base G to A at 415bp in the coding region,resulting in the encoded amino acid No.139 from alanine(Ala)to threonine(Thr).The gene DNA was 1696 bp in length,containing two exons and one intron,the CDS was 519 bp in length and encodes a protein containing 172 amino acids.According to website forecast,the N-terminal of the protein contains 28 amino acids composition of a signal peptide of chloroplast and contains ANK(Ankyrin repeat motif)repeats consisting of 33 amino acid residues at positions 114-146,which belonging to the ankyrin family.520YS protein was found in higher plants,green algae and moss Phylogenetic tree analysis showed that 520YS was more closely related to monocotyledonous plants such as sorghum and maize,and the homology of protein sequence was as high as 77%.(6)Transgenic functional validation:the transgenic expression vector pC2300-actin-520YS,was constructed by wild-type gene and transformed into the mutant by Agrobacterium-mediated transformation.The positive transgenic plants were green and the photosynthetic pigment content was found to be normal wild type.Suggesting that the yellow-green leaf mutant phenotype was caused by mutations in the 520YS gene.(7)Expression analysis of the gene 520YS:RT-PCR was used to detect the expression pattern of 520YS gene at seedling stage and booting stage.The results represent that the 520YS gene was expressed in leaves,leaf sheaths,spikelets and stems,but not expressed in roots.Indicating that the gene is expressed barely in green tissue.(8)Subcellular localization:the 520YS gene was ligated into the pC2300-35S-eGFP expression vector and transferred into rice protoplasts by PEG.The results showed that the 520YS protein was located on the chloroplast by laser scanning confocal microscope.2.Genetic analysis and fine mapping of dwarf mutant d6218 in ricePlant height as an important agronomic trait of rice plant type,which is closely related to photosynthetic efficiency,lodging resistance and yield,is one of the main factors affecting rice yield.Therefore,the study of molecular mechanism of rice plant height has a very important theoretical significance for rice breeding.In this study,the dwarf mutant d6218 was isolated from the F2 generation of 178R/LP213 hybrid,the phenotypic identification,genetic analysis and gene mapping of the dwarf mutant were used to lay the foundation for further screening and functional verification of candidate genes.(1)Mutant phenotype observation and agronomic traits analysis:d6218 isolated from heterozygous parents,seedling no significant difference and in the growth period of 62 days or so gradually dwarf traits,mutant to mature stage plant height less than half of the normal plant,leaves shortened,narrow and erect and darkened in color,d6218 also showed fertility decreased or even infertile.(2)Genetic analysis and gene mapping of mutants:genetic analysis showed that d6218 dwarf mutant trait was regulated by a pair of recessive nucldar genes.The d6218 was fine-coded in the range of 88.0 kb at the end of the 10th chromosome by molecular markers,between the SSR markers X13 and RM6673,with the genetic distance of 0.28 cM and 0.14 cM,respectively.The range has not yet been reported as dwarfing genes,and we consider it to be a new dwarf gene,temporarily named d6218. |
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