Cloning Of CDNA Encoding For Associated Genes Of Flavone Biosynthesis In Chinese White Olive (Canarium Album Raeusch. Cv. Changying) Leaves

In the present study, the length cDNA of cinnamate-4-hydroxylase (C4H) , cDNA fragment of Phenylalanine ammonia-lyase (PAL) and 4-coumarate CoA: ligase(4CL) from Chinese white olive leaves were cloned with RT-PCR and RACE, which set up a foundation for future study of the relationship between these genes and Falconoid Biosynthesis in molecular level, for revealing of the molecular mechanism of Falconoid Biosynthesis and falconoid exploitation in Olive Leaf .The results were as follows:1 Cloning of the full length DNA of flavanone cinnamate-4-hydroxylase(C4H) by RT-PCR and RACEThe full-length cDNA of Chinese white olive C4H is 1819 bp. It contains an ORF encoding 505 amino acids(Mw=57.837 kDa, pI=9.72 ),a 5′untranslated region of 63 bp and a 3′flanking sequence of 238 bp. Compared with C4H gene of others plants by Blast software, Chinese white olive C4H cDNA respectively had 82 % , 82 %, 81 % , 80 % and 81 % identity with that of Populus kitakamiensis superfamily, Citrus x paradisi, Gossypium arboreum, Rubus coreanus, and Camellia sinensis C4H genes. The comparison among the putative amino acid sequence with Populus kitakamiensis superfamily C4H and Citrus x paradisi C4H and Gossypium arboreum C4H showed that identities were 93 % and 92.7 % and 92.7%, respectively. It had the lowest identity with that of Arabidopsis thaliana C4H, namely 81%. This cDNA had been registered in GeneBank with accession number FJ821504. Conserved Domain Search revealed that Chinese white olive C4H was only one complete domain of P450 superfamily, which meaned that C4H protein was a cytochrome protein.2 Cloning of cDNA frament of Phenylalanine ammonia-lyase (PAL) by RT-PCR and RACEA cDNA fragment of 2172 bp was obtained. This cDNA had been registered in GeneBank with accession number FJ821505. Compared with PALs of other plants, it showed that the deduced Chinese white olive PAL gene had high similarity with others plants PALs by Blast software. Conserved Domain Search revealed that PAL was composed of three domains, PAL-HAL, Lyase_I_like surperfamily and PAL. In this three regions each has a dehydrogen acyl-alanyl electrophilic center. These similar structure regions were close to their property and function.3 Cloning of cDNA frament of 4-coumarate:CoA ligase(4CL) by RT-PCR and RACEA cDNA fragment of 360 bp was obtained. Compared with 4CLs of other plants, the deduced Chinese white olive PAL gene had high similarity with Populus kitakamiensis , Eriobotrya japonica , Nicotiana tabacum , Nothofagus pumilio by Blast software, the identities were more than 80% except Arabidopsis thaliana.and Capsicum annuum. This cDNA had been registered in GenBank with accession number FJ821503. Conserved Domain Search revealed that cDNA frament of deduced 4CL contained an malformed conserved domain, LuxE superfamily. LuxE catalyses the formation of an acyl-protein thioester from a fatty acid and a protein, a well known catalyses which played in a role as an activation in acyl reduction.Above all, C4H, PAL and 4CL genes might play an important role in flavonoid synthesis of the Chinese white olive leaves. The cloning of these three genes will contribute to further development and utilization of the flavonoids in Chinese white olive leaves.