Cloning And Primary Characterization Of DFR In Iris Laevigata

Flower color is the most important ornamental trait of flower germplasm resources,and it is also one of the most important target traits of flower germplasm innovation.Understanding the color formation mechanism of different flower colors of different species can help to achieve color regulation through modern molecular biology methods,obtain new varieties with special colors and enhance ornamental value.Special flower color engineering breeding is also the focus of Iris plant breeding research.Iris laevigata is a rare cold-tolerant aquatic perennial ornamental flower in northern China,with blue and large flowers.The flower of Iris laevigata is beautiful and strange,but the color is relatively simple.The discovery of the Iris laevigata var.alba which flower is white under the natural population provides a very good control material for the study of the mechanism of flower color production of Iris laevigata,which is beneficial to open the iris plant and expand the research on the color metabolism of monocotyledon.Dihydroflavonol 4-reductase(DFR)is an important enzyme in the synthesis of anthocyanins in plants.Based on the high-throughput transcriptome data analysis of blue and white flower buds,the DFR gene,a key gene for differential expression of two colors in the anthocyanin metabolic pathway,was significantly down-regulated in white flowers.In this study,the DFR gene was successfully cloned from Iris laevigata and named as IlDFR.The basic characteristics of IlDFR gene alignment,evolution analysis and subcellular localization were analyzed.The expression pattern of IlDFR gene was analyzed by Real-time PCR.The function of IlDFR gene was introduced into the model tobacco.The main findings are as follows:1.The full-length sequence of the IlDFR gene was cloned from the petals of Iris laevigata.Sequence analysis of the target gene revealed that the open reading frame(ORF)of the IlDFR gene is 1026 bp in length and encodes 341 amino acids.The results of q-PCR showed that the IlDFR gene was mainly expressed in the flower of Iris laevigata and Iris laevigata var.alba,and the expression level in the leaves was low.At different stages of flowering,the expression of IlDFR gene in the petals of Iris laevigata and Iris laeivigafa var.alba reached the maximum at the flowering stage(S4),but the expression level in the flower of Iris IwvigcUa var.alba was lower than that of Iris laevigata.The expression level of IlDFR gene in the anther of Iris laevigata var.alba was higher than that in the anther of Iris laevigata.2.Bioinformatics analysis results show that IlDFR protein may be a hydrophilic protein,no signal peptide,with serine,threonine,tyrosine phosphorylation sites;?-helix and random coil is the main two of IlDFR protein hierarchical structural elements.Using the Blastp prediction of NCBI,the amino acid sequence encoded by IIDFR has a typical DFR protein functional domain and belongs to the NADB-Rossmann superfamily.Phylogenetic analysis showed that the protein encoded by the IIDFR gene had higher homology with carrots,followed by potatoes.3.The pBI121-IlDFR plant overexpression vector and pBI121-IlDFR-GFP plant expression vector were constructed by double restriction enzyme digestion,and transferred into tobacco by Agrobacterium-mediated transformation.The transgenic tobacco DNA was identified by PCR and the identified transgenic tobacco was extracted.Q-PCR analysis showed that the exogenous IIDFR gene has been successfully transferred into the tobacco genome and expressed.Subcellular localization analysis of the IlDFR gene by gene gun method revealed that DFR protein acts in the cytoplasm and cell wall.4.According to the results of q-PCR,three transgenic lines with high expression of IIDFR gene were selected:IlDFR-1,I1DFR-3 and IlDFR-8.The phenotype was compared with wild type tobacco,and it was found that the tobacco was transferred to IIDFR gene.The color is darker,dark pink(RHS 58C),the edge of the petal is curled;the flower diameter,flower length,male and pistil length are smaller than that of wild type tobacco,and the ovary diameter is larger than wild type tobacco.The difference of IIDFR-1 was the most obvious,followed by IIDFR-3 and IlDFR-8.The number of flowers at the top of the main branch of transgenic tobacco(23-33)was higher than that at the top of wild-type tobacco(13-18)and the difference is obvious.It is speculated that the IIDFR gene of Iris laevigata is involved in the regulation of quantitative traits of flower color and flower morphology.