Analysis Of WD40 Gene Family In Lettuce And Fine Gene Mapping Of Controlling Red Leaf In Amaranth

In this study,the WD40 family in lettuce genome was systematically analyzed,and179 WD40 transcription factors were found in the lettuce genome.Among them,RLL4belongs to Group I and Ls TTG1 belongs to Group III.Evolutionary analysis indicates that these two genes may have functional differentiation.Transgenic results show that Ls TTG1 promotes anthocyanin synthesis,while RLL4 negatively regulates anthocyanin synthesis.Compared with control plants,396 common differentially expressed genes（DEGs）were screened from RLL4 overexpression plants and RLL4 editing plants.The RNA-seq data showed that RLL4 gene can regulate the expression of PAL,CHS,CHI,F3H,DFR,ANS and GST,which are the structural genes of the anthocyanin pathway.The results of yeast one-hybrid assay showed that RLL4 directly regulated the expression of Ls MYB12 transcription factors,and the results of yeast two-hybrid assay showed that RLL4 and Ls MYB12 could interact weakly,while Ls MYB12 directly activated the expression of CHI,DFR and ANS,which indicated that RLL4 could not only directly inhibit the expression of Ls MYB12,also can and Ls MYB12 formed after the complex,inhibited the synthesis of anthocyanins.Ls TTG1 knockout plants had 261 DEGs.The functional analysis DEGs of anthocyanin synthesis pathway showed that afte Ls TTG1gene was knocked out,the expression of other genes of anthocyanin synthesis pathway was down-regulated except PAL and CHI,LUC assay showed that Ls TTG1 could not directly activate CHS、CHI and F3H expression.Ls TTG1 only screened RLL1（b HLH）from the yeast library of lettuce,and RLL1 interacts with RLL2（MYB）.The previous results showed that Ls TTG1 promoted anthocyanin synthesis through MBW complex.Amaranth leaves contain a unique antioxidant pigment,compared with other leafy vegetables.Although some genes regulating betacyanin biosynthesis in amaranth have been cloned,the mechanism controlling red formation in leaves is still unclear.Using amaranth as parents to construct F₂segregating population,the genes controlling amaranth color were determined by BSR-seq method in the range of 5,036,841 and5,323,729,about 286 kb,respectively.After gene function annotation,only two genes,DODA and CYP76AD1,were found to be associated with leaf color formation in amaranth.The CYP76AD1 gene had a 10bp deletion in amaranth,which resulted in the premature termination of the protein encoded by CYP76AD1,the DODA gene had five base changes in red and green amaranth,resulting in two amino acids.The expression of DODA and CYP76AD1 in red amaranth was higher than that in green amaranth,so both CYP76AD1 and DODA genes might be the candidate genes for controlling red color formation in amaranth leaves.