Fine Mapping Of A Variegated Leaf Gene WLML1 And Functional Analysis Of A Tolerant Gene OsCATB In Rice

There is an important value in theory and application that using forward genetics and reversed genetics methods study mutants on genetic analysis and molecular mechanism in rice,and reveal important functional genes and biological processes in higher plants.In this paper,forward genetics was used to study the fine mapping and gene cloning of a leaf-color mutant,and the function of a gene encoding catalase B was studied using reverse genetics.1.Genetic analysis and mapping of a new white and lesion mimic leaf?wlml1?mutant in rice.Rice?Oryza sativa?leaf color mutants are ideal materials for studying the mechanism of chlorophyll metabolism and chloroplast development.Exploring rice leaf color mutants has become an important direction for rice functional genomics research.In this paper,a stable genetic mutant with white and lesion mimic leaf selected from the EMS mutant library of indica rice TN1?Taichung Native1?,and it was provisionally named wlml1?white and lesion mimic leaf 1?.A preliminary study was conducted in this mutant including phenotype identification,fine mapping and cloning of the gene.The main results are as follows:1)The mutant showed macular or white spots after four leaf stage,with occasional lesion mimics;white spot at the filling stage,and numerous lesion mimics at the late stage of ripening;transmission electron microscopy revealed that the number of chloroplasts in mesophyll cells at the phenotypic site decreased and the degradation was serious.2)The pigment content and photosynthetic rate of leaves in the wlml1 mutant were measured and results showed that both of them were decrease.Real-time quantitative PCR detected that and genes related to chlorophyll metabolism and chloroplast development were up-or down-regulated.3)Temperature treatment revealed that low temperature accelerated leaves yellowing of wlml1mutant and also induced lesion mimics appearing early.4)WLML1 was mapped on the chromosome 4 between the markers P21 and P23 within the interval of 41-kb genomic DNA region by using F₂ population derived from hybridization between wlml1 mutant and TN1.And there were 9 ORFs in this region.5)Gene sequencing revealed that there was one base mutation in ORF5,which caused an amino acid changes;subcellular localization showed that the gene was located in the chloroplast,and the gene was guessed as the target gene.And we had completed the relevant vector construction and are verifying whether it is the target gene by transgenic technology.2.Function analysis of root length and stress related gene OsCATB in rice.Root growth is one of the important goals for improving grain production.And the active root system can absorb nutrients more efficiently,and it is a key way to improve grain production.Based on chromosome 6 multiple QTL section analysis,genome sequence and expression analysis,the LOC_Os06g51159 was identified as a candidate gene.This gene encoded a catalase isozyme B?OsCATB?,and may be related to the development of root.The homozygous mutants were obtained by using CRISPR/CAS9 technology to study of its function.1.Phenotypic statistics revealed that there were no changes in root length and plant height of OsCATB knockout mutants;physiological data showed no significant changes in H₂O₂ content,ABA content,and CAT activity in the roots,stems,and leaves of mutant.2.It was found that there were differences in OsCATB expression between roots,stems and leaves by real-time quantitative PCR.3.Nitrogen treatment showed that the root length of the mutants was shorter than that of the wild-type plants under the treatment of ammonia nitrogen and nitrate nitrogen.