Identification Of Perianth Color Components,and Cloning And Expressive Pattern Of IlF3’H,IlF3’5’H Genes Of Iris Laevigata

This research was completed under the National Natural Science Foundation of China(31670344).Flower color is one of the important ornamental traits of plants,and blue flower is favored by people.Due to the lack of species of blue flower plants,blue flower breeding has attracted much attention.Iris laevigata is an aquatic blue flower species with strong cold resistance in the genus Iris.The flower color is gorgeous and has high ornamental value.There are few reports on the mechanism of the color formation of its flower color.In this study,I.laevigata as the research object,through the analysis of the main metabolite components of the perianth pieces,to analyze the material basis of the formation of I.laevigata.F3’H and F3’5’H,two important structural genes for flower color regulation,were screened and cloned based on the transcriptome data of I.laevigata.The expression characteristics of F3’H and F3’5’H genes during flowering and seedling stress were detected.The plant over-expression vectors pBI121-IlF3’H and pBI121-IIF3’5’H were successfully constructed,which laid a foundation for engineering breeding of flower color and resistance genes.The main research results are as follows:1.A total of 11 species of 856 species were identified in the perianth tablets of I.laevigata by metabonomics detection.The number of flavonoid compounds was the most,and the number of tannins,lignans and coumarins was the least.Flavonoid compounds are mainly composed of flavonoids,dihydroflavones,dihydroflavanols,isoflavones,flavanols,anthocyanins,flavonoid glycosides and dihydroisoflavones.It was found that anthocyanins were the main chromatite in the perianth of I.laevigata.There were 35 anthocyanins in the perianth of I.laevigata,including 6 kinds of pigments including cornflower pigments,geranium pigments,delphinium pigments,peony pigments,morning glory pigments and mallow pigments.The highest content was delphinin-3-O-glucoside,followed by cornflower pigments,and the least content was geranium pigments.According to the analysis of the floral and color metabolism pathway,it was found that the precursor metabolites of delphinin and cornflower pigment were dihydromyricetin and dihydroquercetin,respectively,which proved that the structural genes F3’H and F3’5’H both played an important role in the color formation of anthocyanins.2.The ORF sequence of IIF3’H and IIF3’5’H gene was cloned from the cDNA of the perianth of I.laevigata,with the length of 1107bp and 918bp,respectively.Bioinformatics analysis showed that both of them were hydrophobic proteins without signal peptides.The former was mainly modified by threonine acidification,while the latter was mainly modified by serine phosphorylation and threonine acidification.Both IlF3’H proteins were mainly composed of a-helix and irregular coils,and IlF3 ’H protein had 20G-FeII_Oxy binding site as well as PcbC oxygenase site.Homology comparison showed that the protein homology of IlF3’H and Lilium speciosum was high.IlF3’5’H protein has high homology with Ensete ventricosum protein,and IIF3’5’H protein has P450 binding site and PLN02687 oxygenase family site.IlF3’5’H protein is a member of P450 supergene family.It may also be related to the synthesis,transport and catabolism of secondary metabolites of cytochrome.3.q-PCR results showed that the expression levels of IlF3’H and IIF3’5’H genes in I.laevigatas were higher than those in leaves.At flowering stage,the expression levels of IIF3’H and IIF3’5’H genes increased firstly and then decreased,and the expression levels of IIF3’5 ’H genes were higher at full florescence(S4)and middle florescence(S3).4.The expression patterns of IlF3’H and IIF3’5’H genes under low temperature stress,salt stress and drought stress were quantitatively detected by real-time fluorescence.The results showed that under low temperature stress,IlF3’5’H gene expression was higher in leaves,and its response expression was mainly in leaves.However,IlF3’H mainly occurred in roots and was significantly expressed with the increase of stress.Under drought stress,the expression levels of IlF3’H and IlF3’5’H in roots were higher than those in leaves,and both of them showed a trend of increasing at first and then decreasing,suggesting that IIF3 ’H and IlF3’5’H could be up-regulated in response to drought and low temperature.Under salt stress,the expression of IlF3’H in young roots was much higher than that in young leaves,and the up-regulated expression of IIF3 ’H in young roots showed an obvious and strong stress response mechanism.However,the expression of IlF3 ’5’H did not change much and only responded to the resistance to salt stress to some extent.5.The two gene fragments were linked to pBI121 respectively by adding XbaI and BamHI to IlF3’H and IlF3’5’H by double enzyme digestion method.Finally,the plant overexpression vectors pBI121-IlF3’H and pBI121-IlF3’5’H were successfully constructed.