Cloning and expression analysis of ImLHY and ImC4H genes in Impatiens morsei Hook. f.

【Objective】 The purpose of the study was to clone and analyze the expression of the late hypocotyl protein gene（ImLHY）and cinnamic acid-4-hydroxylase gene（ImC4H）of Impatiens morsei Hook. f.，to explore the color dif ference between variegation and non-variegation areas in vexil and wing petals of I. morsei，and to clone LHY and C4H genes，so as to provide a theoretical basis for molecular regulation mechanism of flower variegation，flower color im provement and new variety breeding of I. morsei.【Method】I. morsei was used as the material，the colorimetric values， contents of total anthocyanins and total flavonoids of variegation and non-variegation areas in vexil and wing petals were determined. The ImLHY and ImC4H genes were cloned，and their sequence characteristics，protein structure and phylogeny were analyzed by biological software. Real-time fluorescence quantitative PCR was used to detect the expression patterns of ImLHY and ImC4H genes at different developmental key stages（bud stage，blooming stage and decay stage）and in different pigmented regions of vexil and wing petals.【Result】The a^* value（redness value）and C^* value（chroma value）of the variegation area were higher than those of the non-variegation area in vexil and wing petals，and L^*（brightness value）of the non-variegation area was greater than that of the variegation area. The b^* value（yellowness value）of the variegation area was higher than that of the non-variegation area in vexil，while the b^* value（yellowness value）of the variegation area was less different from that of the non-variegation area in wing petal. The contents of total anthocyanins and flavonoids in vexil and wing petals were higher than those in non-variegation area. The full-length cDNA of ImLHY and ImC4H genes obtained from cloning were 1698 and 1518 bp，encoding 565 and 505 amino acid residues respectively. The GenBank accession numbers were OR096417 and OR096417. ImLHY and ImC4H proteins were hydrophilic unstable proteins without transmembrane domains. ImLHY protein had a Myb DNA-binding conserved structural domain and a SANT structural domain of the MYB transcription factor family，which was localized in the nucleus. ImC4H protein contained a P450 superfamily structural domain（PLN02394），and was located in the endoplasmic reticulum. The amino acid sequences of the ImLHY and ImC4H proteins had 68% and 94% similarity with Impatiens glandulifera. In phylogenetic evolutionary tree，both of them were clustered together with LHY and C4H of I. glandulifera respectively. The two genes were expressed in the three key stages of flower development（bud stage，blooming stage and decay stage）and in the variegation and non-variegation areas of vexil and wing petals，and the relative expression was higher at the blooming stage overall. At blooming stage，the relative expression of ImLHY and ImC4H genes was significantly higher in variegation area of vexil than in non-variegation area（P<0.05），and higher in non-variegation area of the wing petals than in variegation area.【Conclusion】ImLHY and ImC4H genes mainly regulate the formation of variegation in vexil during the blooming stage， and mainly regulate the formation of non-variegation area in wing petals. It is speculated that there are different regulatory mechanisms for the formation of variegation in vexil and wing petals.