The Cloning Of Pomelo Fruit Flavonoids Synthesis Genes CDNA And Bioinformatics Analysis

In the present study, the length cDNA of flavanone-3-hydroxylase (F3H), cDNA fragment of chalcone isomerase (CHI), chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL), from Citrus grandis cv.Guanximiyou 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 pomelo fruit. The results were as follows:1 Cloning of the full length DNA of flavanone-3-hydroxylase (F3H) by RT-PCR and RACEThe full-length cDNA of Citrus grandis cv.Guanximiyou F3H is 1302 bp. It contains an ORF encoding 362 amino acids (Mw=40.497 kDa, pI=5.97 ). Compared with F3H gene of others plants by Blast, Citrus grandis cv.Guanxi pomelo F3H cDNA respectively had 97%,87%,86%,85% and 84% identity with that of Citrus sinensis, Dimocarpus longan, Gossypium hirsutum, Gossypium barbadense and Vitis vinifera F3H genes. The comparison among the putative amino acid sequence of Citrus sinensis superfamily F3H and Dimocarpus longan F3H and Gossypium hirsutum F3H showed that identities were 93 % and 92.7 % and 92.7%, respectively. It had the lowest identity with that of Arabidopsis thaliana F3H, namely 81%. This cDNA had been registered in GenBank with accession number GQ121373. Conserved Domain Search revealed that Citrus grandis cv.Guanximiyou F3H was only one complete domain of 20G-FeII-Oxy superfamily, which meaned that F3H protein was an oxygenase protein.2 Cloning of cDNA frament of chalcone isomerase (CHI) by RT-PCR and RACEA cDNA fragment of 782 bp was obtained. This cDNA had been registered in GenBank with accession number FJ976039. Compared with CHI gene of others plants by Blast, Citrus grandis cv.Guanximiyou CHI cDNA respectively had 96%,95% identity with that of Citrus sinensis, Citrus reticulata and CHI genes. Conserved domain search revealed that CHI was composed of one domain Chalcone superfamily. The function of domain catalyzes the reaction from Chalcone to Naringenin.3 Cloning of cDNA frament of Chalcone synthase (CHS) by RT-PCRA cDNA fragment of 280 bp was obtained. Compared with CHS of other plants, the deduced Citrus grandis cv.Guanximiyou CHS gene had high similarity with Camelliasinensis Oltze by Blast.Conserved domain search revealed that cDNA frament of deduced CHS contained a cond-enzymes conserved domain, LuxE superfamily. LuxE CHS catalyzes the reaction, 4-coumaroyl-CoA and Malonyl-CoA to Chalcone 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 activation in acyl reduction.4 Cloning of cDNA frament of Phenylalanine ammonia-lyase (PAL) by RT-PCRA cDNA fragment of 875 bp was obtained. Compared with PALof other plants, it showed that the deduced Citrus grandis cv.Guanximiyou PAL gene had high similarity with others plants PALs by Blast. Conserved domain search revealed that PAL one consevered domain, PAL-HAL. This region has a dehydrogen acyl-alanyl electrophilic center.This similar structure region was closed to the property and function of PAL.Above all, F3H, CHI, CHS and PAL genes might play an important role in flavonoid synthesis of the Citrus grandis cv.Guanximiyou fruit. The cloning of these four genes will contribute to further development and utilization of the flavonoids in Citrus grandis cv.Guanximiyou.