Cloning And Expression Analysis Of Flavonoid-3’,5’-hydroxylase Genes From Three Wild Ornamental Plants In Yunnan

Flower color is one of the most important ornamental and economic traits. Blue can represent elegance and the feeling of dream. However, the four-major cut flowers in the world have little blue-flowering varieties. Therefore, to breed blue flower has become the focus in flower color modification. Lack of blue flowers is mainly due to the inactivity of flavonoid-3’,5’-hydroxylase (F3’5’H), especially in rose, carnation and lily. F3’5’H is the key enzyme for delphinidin synthesis, which is generally required for the expression of blue or purple flower color. F3’5’H genes have been isolated from many plants, and have also been used in flower breeding abroad. The novel blue-hued roses have been successfully marketed, conferring genetic engineering of blue flower universal attention. In contrast, study related to blue flower breeding is paid little attention in China. In fact, we have rich blue flower resources, which can be utilized to explore blue flower genes for further application in genetic engineering.In the present study, we got the cDNA sequences of F3’5’H gene from Aconitum carmichaeli Debx., Aconitum vilmorinianum and Platycodon grandiflorus A. CD., and analyzed their temporal and spatial expression patterns in various tissues. The results were as followed:1. According to the known F3’5’H amino acid sequence, several degenerate primers were designed. Using RT-PCR and RACE approaches, the novel F3’5’H genes, named Ac-F3’5’H (GenBank:JN635708) and Pg-F3’5’H (GenBank:JQ403611) were cloned from Aconitum carmichaeli Debx. and Platycodon grandiflorus A. CD., respectively. The cDNA sequence including a whole open reading frame of Ac-F3’5’H was1720bp and encoded506amino acids; The cDNA sequence including a whole open reading frame of Pg-F3’5’H was1787bp and encoded532amino acids. Using the amplification primer of Ac-F3’5’H, a novel F3’5’H gene, named Av-F3’5’H (GenBank:JQ806761), was cloned from Aconitum vilmorinianum. The cDNA sequence was1563bp, and included a whole open reading frame, which encoded506amino acids.2. Sequence alignment showed that the amino acid sequence encoded by these three F3’5’H genes had high homologous with the F3’5’H from other plants. They belonged to P450family, and had three motifs:"PPGP" motif, I-helix, and P450haem-binding. Homology comparison and dendrogram analysis showde that Av-F3’5’H and Ac-F3’5’H had95%similarity, and had88%similarity with F3’5’H form Delphinium grandiflorum. Pg-F3’5’H have80%similarity with F3’5’H form Campanula medium. The result showed the evolutional relationship of F3’5’H, which met the plant taxonomy.3. The temporal and spatial expression patterns of these three genes:Ac-F3’5’H, Av-F3’5’H, and Pg-F3’5’H were analyzed. Semi-quantitative RT-PCR analysis showde that the F3’5’H genes were highly transcribed in middle and late phase of flower development, which was concomitant with the appearance of anthocyanins in flower tissue. The transcripts of F3’5’H kept increasing during flowering. Ac-F3’5’H and Pg-F3’5’H exhibited the highest transcription in the fourth stage, while Av-F3’5’H in the third stage. F3’5’H showed a tissue-specific pattern, The transcripts were abundant in the purple flowers but absent in leaves or roots.This indicated that the F3’5’H were developmentally regulated and might play a crucial role in regulating the forming of blue flowers.4. Using Ac-F3’5’H, we constructed a vector pCAMBIA1301-220.6-Ac-F3’5’H for plant transformation.