Analysis Of Flavonoids In Different Tissues Of Daylily And Study On Function Of F3H Gene

Daylily（Hemerocallis spp.）,a kind of perennial herbaceous plant in the family aphrodiaceae,combines ornamental,medicinal,and edible values with extremely high application value.The content of anthocyanins,anthocyanin glycosides,and total flavonoids in different tissues and developmental stages of daylily’Black-eyed Stella’was determined in this study.The expression patterns of genes related to total flavonoid and anthocyanin synthesis pathways was analyzed through transcriptome sequencing.The expression pattern of flavanone-3-hydroxylase（Hf F3H）gene in different developmental stages of tissues was analyzed by using fluorescence quantitative PCR（q RT-PCR）technology.A CRISPR/Cas9gene editing vector driven by the high expression promoter of ovum cells and proliferating cells was generated for the Hf F3H gene based on daylily genome,transcriptome data,bioinformatics analysis.These results provided a basis for exploring the function of the F3H gene in regulating total flavonoid and anthocyanin biosynthesis.The main research results are as follows:（1）The content of anthocyanins and total flavonoids in different tissues of daylily’Black-eyed Stella’at different developmental stages was analyzed and determined.The results showed that the content of epidermal anthocyanins was the highest,with a content of 35.39U/g;followed by flower eyes,with a content of 31.87 U/g;and root had the lowest content,only 6.61 U/g.Total flavonoids were highest in large flower buds,with a content of 33.07mg/g,and lowest in flower stalks,with a content of 0.17 mg/g.（2）Using different tissues of daylily’Black-eyed Stella’at different stages as materials,transcriptome sequencing（RNA-Seq）was performed,and 87,615 Unigenes with an average length of 969.55 bp were obtained by assembly and splicing.Differential genes involved in the biosynthesis pathways of total flavonoids and anthocyanins were screened through transcriptome data analysis,and 8 key enzyme coding genes that may participate in the biosynthesis of total flavonoids and anthocyanins were further analyzed and identified through data screening,including Hf F3H,Hf BZ1,Hf CHS3,Hf CHS2,Hf4CL,Hf ANS,Hf PGT1,and Hf FLS."（3）The flavanone-3-hydroxylase gene was isolated from the’Black-eyed Strela’Scutellaria baicalensis and named Hf F3H.Bioinformatics analysis showed that the Hf F3H gene belongs to the PLN02515 superfamily.The amino acid structure,physicochemical properties and functions of the Hf F3H gene were predicted and analyzed.The Hf F3H gene had 376 amino acids,encoded a protein molecule with a molecular formula of C₁₈₆₆H₂₉₄₂N₅₀₄O₅₆₉S₁₇,a molecular weight of 42085.94,and a theoretical isoelectric point of5.16.The secondary structure of the Hf F3H gene protein was mainly composed ofα-helix,β-turn,extended chain and irregular coil,with irregular coil as the main part.The Hf F3H gene-encoded protein did not have a transmembrane region,and the amino acid sequence belonged to a hydrophobic protein,with many phosphorylation sites and no signal peptide.The probability that Hf F3H was located in the cytoplasm was 47.8%,and it was closely related to the F3H gene of Sophora flavescens.（4）To investigate the role of Hf F3H in the biosynthesis pathways of total flavonoids and anthocyanins in daylily,the full-length sequences of c DNA and genome of Hf F3H were analyzed to figure out the sequences of intron and exon.The results here showed that Hf F3H gene contains three exons and two introns,which validated by genomic DNA amplification followed by sequencing.Two target sites for gene editing were designed on the first and second exons of the Hf F3H gene,respectively.The gene editing vectors,p XEE-Zm Ubq-Cas9-Hf F3H and p XEE-ECIfp-Cas9-Hf F3H,were genertaed.The gene editing experiments will be performed by pollen-tube pathway transformation,and the taget cells for transformation is egg cell or other germ cells.Therefore,Cas9 gene of gene editing vectors was controlled by a monocotyledonous plant constitutive promoter（Zm Ubq）which highly expressed in proliferating cells or an egg specific promoter（ECIfp）,and the guide RNA was controlled by the monocotyledonous plant constitutive promoter,rice U3 and U6promoters.The vectors were named p XEE-EC1fp-Cas9-Hf F3H and p XEE-Zm Ubq-Cas9-Hf F3H,laying the foundation for further study of the function of the Hf F3H gene.