Function Characterization Of Rec Gene Family In Arabidopsis And Genetic Cloning Of Gene That Promotes The Accumulation Of Anthocyanin In Radish Root

This study is composed of two parts:function characterization of REC gene family in arabidopsis and genetic cloning of gene that promotes the accumulation of anthocyanin in the epidermal layer of the swollen part of the radish root.The first part is about the generation of knock-out mutants of REC gene family which is required to the mechanisms of establish the cellular volume devoted to all chloroplasts and phenotype identification of these mutants.Chloroplast plays a fundamental role in yield and quality of crop which was determined by the capacity of photosynthesis regardless of the influence of environment.the Maximum capacity of photosynthesis was determined by the number and cellular volume devoted to all chloroplast in the mesophyll cell.Plastid division affects the number of chloroplast in mesophyll cell.REC gene family is associated with the mechanism of establish the cellular volume devoted to all chloroplasts.We found that a new rec mutant which has a different insertion of T-DNA with the mutants published before has a stronger chlorophyll deficiency.Thus we proposed that the function of REC gene family was underestimated in this mechanism.We generated knock-out rec mutants with CRISPR/Cas9 technology.In addition,the chlorophyll level is lower than before in knock-out rec1 mutants.There are also less chloroplast plan area in knock-out rec1 mutants.However,the chloroplast number is not as many as rec1 mutants which has T-DNA insertion.The result indicates REC1 is independent of chloroplast division.Furthermore,the chlorophyll deficiency phenotype of T₁ plant of rec2 friendly mutant generated by CRISPR/Cas9 method indicates the underestimated function of REC gene family could come from REC2.The second part is about the genetic cloning of a gene that promotes the accumulation of anthocyanin in the epidermal layer of the swollen part of the radish root.The ratio of purple and white skin of radish root is 3:1 that indicates the mapping population is segregating a single allele.Thus we perform a BSA and RNAseq using white and purple epidermal layer from the swollen part of the root of the radish and analysis the differential expression genes of two pools.The preliminary mapping result shows that the interest gene is located on chromosome 7,from 5.680 MB to 6.393 MB depend on the analysis of linked marker.Due to the low quality of chromosome assembly of this region,we can't minimize the scale of candidate region depend on the marker in candidate region.There are 64 genes which was annotated in this region depending on the reference genome.There are two possible candidate gene in this region:one of which encodes protein with a MYB domain.The another candidate gene encodes a HY5transcription factor which could positively regulates the anthocyanin biosynthesis in Arabidopsis.There is no difference in the promoter and coding sequence of Rs-HY5.But there are 17 SNP in Rs-MYB,two of which could transform the amino acid from Methionine to Valine.