| In the present study,the full length cDNA of flavanone 3-hydroxylase(F3H)and cDNA fragment of Fructokinase(FRK)from longan(Dimdcarpus longana Lour.) embryo were cloned by RT-PCR and RACE.The recombinant prokaryotic expression vector pET-29a inserted F3H gene was constructed and attempted to express in E.coli BL21.It set up a foundation for discovering the molecular inbeing of development and differentiation in longan embryo as well as adopting gene engineering technology to cultivate high quality fruit.1 Cloning of the full length cDNA of flavanone 3-hydroxylase(F3H)by RT-PCR and RACEThe full-length cDNA of longan F3H is 1404 nucleotides long,including an ORF encoding 365 amino acids(Mw=41.112kDa,pI=5.55),a 5' untranslated region of 119 bp and a 3' flanking sequence of 118 bp.It was concluded that longan F3His a hydrophilic protein.When the nucleotide sequence compared with Gossypium hirsutum F3H gene and Citrus sinensis F3H gene and Fragaria x ananassa F3H gene, it showed that identity were 84%and 82%and 83%,respectively.The comparison between the putative amino acid sequence with Gossypium hirsutum F3H and Citrus sinensis F3H and Vitis vinifera F3H showed that identity were 90.76%and 89.04% and 88.49%,respectively.This cDNA has been registered in GenBank with accession number EF468104.2 Expression of longan F3H proteins in E.coli.The longan F3H gene cDNA was inserted into the vector pET-29a.The expression construct was transformed into BL21 and was induced.The result showed that longan F3H gene has failed to express.It was concluded that the expression vector and host were not suitable for expression of longan F3H.3 Cloning of cDNA fragment of Fructokinase(FRK)by RT-PCR and RACEA 850 bp cDNA fragment was obtained.Compared with FRKs of other plants, longan FRK gene cDNA had 87%,81%,75%identity,with that of Citrus sinensis, lycopersicon esculenturn and Arabidopsis thaliana FRK genes,respectively. This cDNA sequence has been registered in GenBank with accession number EU600093. |
