Molecular Cloning And Expression Analysis Of An HmDFR Gene In Hydrangea Macrophylla

Hydrangea macrophylla is a shrub of the Saxifragaaceae family.Different hydrangea cultivars have different colors and patterns,which were widely used in garden cultivation,landscape design and fresh cut flower materials because of the ornamental value.Flower color is one of the most important characteristics of ornamental plants.Clarifying flower-color-related regulatory pathways has important theoretical significance for breeding new cultivars.In order to explore the formation mechanism of different colors of Hydrangea,we completed the transcriptome sequencing of three different sepals colors of Hydrangea Acccoding to the transcriptome analysis,We found that the expression abundance of dihydroflavonol-4-reductase gene family in Hydrangea is closely related to flower color.The transcript number Cluster-33435.81339 showed a significant positive correlation with color shade,We cloned the full-length sequence of this transcript,which confirmed that it encodes a dihydroflavonol-4-reductase in Hydrangea,and it was named as HmDFR The expression patterns of this gene in roots,stems,leaves,buds and sepals at different developmental stages of different Hydrangea were analyzed by real-time quantitative PCR,at the same time,we determined the anthocyanins content of sepals to explore the function of HmDFR gene in the formation of flower color of different Hydrangea cultivars.The main results of this study are as follows:1.Anthocyanins determination in sepals of 'H.macrophylla','Endless summer' and 'Emile Mouillere'.We divided sepals into 6 different developmental stages according to the color depth by observing the color changes of the three Hydrangea cultivars and determined the anthocyanins content.The result showed that the sepals of the H.macrophylla' S1 period are green with anthocyanins content of 464.82 ?g·g-1,S4 period are red with anthocyanins content of 1117.34 ?g·g-1.The sepals of the 'Endless Summer'SI period are light green with anthocyanins content of 388.11 ?g·g-1,S4 period are blue with anthocyanins content of 900.24 ?g·g-1.The sepals of the 'Emile Mouillere' S1 period are light green,S2 and S3 period are white with anthocyanins content between 263.63?g·g-1 and 289.35 ?g·g-1,S4 period are light red with anthocyanins content of 592.73 ?g·g-1.The study have shown that the anthocyanins content of Hydrangea sepals increase with the deepening of the coloration degree,and gradually decrease as the sepals color fades.2.Cloning and identification of HmDFR,The HmDFR gene fragments cloned from sepals of'H.macrophylla','Endless Summer' and 'Emile Mouillere' were the same.The length of HmDFR fragment was 888 bp,and its CDS sequence was 855 bp,which encodes a protein product of 284 amino acids.Bioinformatics analysis showed that HmDFR was conserved in structure and function.3.Analysis of HmDFR gene expression pattern.HmDFR gene has tissue expression specificity in 'H.macrophylla','Endless Summer' and'Emile Mouillere'.The expression level of this gene in sepals shows a trend of 'H.macrophylla'>'Endless Summer'>'Emile Mouillere',and as the sepals color deepens,the expression of HmDFR gene gradually increases,during the S4 period,the sepal color was the highest,so as its expression level,and the gene expression decreases as the sepals color fades.Furthermore,the expression level of HmDFR has a significant positive correlation with the anthocyanins content of sepals,which suggests that HmDFR might play an important role in the growth of sepals.