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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Molecular cloning of flavonoid 3¡¦,5¡¦-hydroxylase cDNA from the petals of Verbena x hybrida

Hung, Cheng-yu 12 July 2004 (has links)
Flavonoid 3¡¦,5¡¦-hydroxylase (F3¡¦,5¡¦H) is the key enzyme that catalyzesthe anthocyanin biosynthesis pathway for the expression of blue or purple flower color. The garden crops of Verbena x hybrida were used to clone its F3¡¦,5¡¦H gene for the investigation of flower color engineering and regulatory program. Degenerate primers were designed from the conservative regions of other published F3¡¦,5¡¦H genes to amplify the expectant DNA fragment . A full-length cDNA of the F3¡¦,5¡¦H gene designated VhFH1 (AY604727) was cloned by the method of 5¡¦and 3¡¦ RACE, and its genomic DNA sequence was isolated by the IPCR strategy. Nucleotide sequence alignment revealed that VhFH1 contains two introns and a 1542 bp open reading frame encoding a polypeptide of 514 amino acid residues, and there could be a promoter sequence with TATA box signal in the upstream of the transcription start site. The amino acid sequence of VhFH1 was compared with the previous reported F3¡¦,5¡¦H and showed between 50¢Mand 77¢Midentity with those species. The expectant molecular mass and isoelectric point of VhFH1 protein is 57 KD and 7.69, respectively. There are three typical motifs of the F3¡¦,5¡¦H that belongs to the cytochrome P450 proteins in the VhFH1 predicted protein. According to the above-mentioned conjecture, VhFH1 is a full-length cDNA of the F3¡¦,5¡¦H gene in the V. hybrida. This cDNA fragment was inserted into the plant expression vector pCambia 1304 and could be detected the expression in E. coli by RT-PCR and protein electrophoresis. It is practicable to transform the horticultural plants with these vectors to create novel flower colors in the future. Furthermore, transcripts of the F3¡¦,5¡¦H gene were detected in the blue, purple and white flowers but not in the red one as revealed by RT-PCR. These results are advantageous in the further investigation of regulatory factors of the anthocyanin biosynthesis pathway in the V. hybrida.

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