The CDNA Cloning And Bioinformatic Analysis Of Flavonoid Biosynthesis-related Genes In Chinese White Olive Leaves

In this research, the length cDNA sequence of flavonoid 3-hydroxylase(F3H), chalcone isomerase(CHI) and cDNA fragment of chalcone synthase(CHS) are cloned by RT-PCR and RACE, which provid the theoretical basis for flavonoid biosynthesis and exploitation in Chinese White Olive. The main results are as follows:1 Cloning and analysis of the full-length cDNA of F3H from Canarium album Raeusch.cv.Changying by RT-PCR and RACEThe full-length cDNA of Chinese White Olive leaves F3H is 1295 bp. It contains an ORF encoding 364 amino acids(Mw=41084.9 Da, pI=5.47), a 5′untranslated region of 41 bp and a 3′flanking sequence of 159 bp. The grand average of hydropathicity (GRAVY) of F3H is -0.437. The instability index (II) is computed to be 42.34. This classifies the protein as unstable. Nucleotide sequence blasting indicates F3H of Chinese White Olive leaves respectively has 84%,84%,83%,83%, 82% and 82% identity with that of Litchi chinensis, Citrus maxima, Dimocarpus longan, Citrus sinensis, Gossypium hirsutum, Gossypium barbadense. Conserved Domain Search revealed that Canarium album Raeusch.cv. Changying F3H was composed of three domains, 20G-FeII-Oxy superfamily, PLN03176 and PLN02515. The holoenzyme has the activity EC: 1.14.11.2 catalysing the reaction: Procollagen L-proline + 2-oxoglutarate + O2 <=> procollagen trans- 4-hydroxy-L-proline + succinate + CO2. 2OG-Fe(II) oxygenase superfamily also includes lysyl hydrolases, isopenicillin synthases and AlkB. This cDNA had been registered in GenBank with accession number JF728822.2 Cloning and analysis of the full-length cDNA of CHI from Canarium album Raeusch.cv.Changying by RT-PCR and RACEThe full-length cDNA of Chinese White Olive leaves CHI is 1054 bp. It contains an ORF encoding 223 amino acids(Mw=24247.9 Da, pI=5.58) , a 5′untranslated region of 50 bp and a 3′flanking sequence of 332 bp. The grand average of hydropathicity (GRAVY) of F3H is 0.013. The instability index (II) is computed to be 34.60. This classifies the protein as stable. Nucleotide sequence blasting indicates CHI of Chinese White Olive leaves respectively has 80%,80%,80%,79% and 78% identity with that of Citrus unshiu, Citrus sinensis, Citrus maxima, Populus trichocarpa, Vitis labrusca. Conserved Domain Search revealed that CHI contains a domain , Chalcone superfamily. Chalcone-flavanone isomerase is a plant enzyme responsible for the isomerisation of chalcone to naringenin, a key step in the biosynthesis of flavonoids. This cDNA had been registered in GenBank with accession number JF728821.3 Cloning and analysis of cDNA fragment of CHS from Canarium album Raeusch.cv.Changying by RT-PCR and RACEA CHS cDNA fragment of 782 bp from Chinese White Olive leaves was obtained, a 5′untranslated region of 117 bp. Nucleotide sequence blasting indicates CHS of Chinese White Olive leaves has the high homology with other plants. Conserved Domain Search revealed that Canarium album Raeusch.cv. Changying CHS contains an malformed domain, cond_enzymes superfamily. Family of enzymes that catalyze a Claisen-like condensation reaction. Members are share strong structural similarity, and are involved in the synthesis and degradation of fatty acids, and the production of polyketides, a diverse group of natural products. This cDNA had been registered in GenBank with accession number JF728822.Followed the research of Wang Yunmei, the length cDNA sequence of flavonoid 3-hydroxylase(F3H), chalcone isomerase(CHI) and cDNA fragment of chalcone synthase(CHS) were cloned in this research, which is helpful to develp flavonoid biosynthesis and expression study in Chinese White Olive, to clear the gene evolution function.