| Daidai (Citrus aurantium), a sour orange variety, belongs to the family rutaceae. It is a kind of woody plant originated in China, the flowers of which are very fragrant. Seville orange is used to process scented tea, extract flavor and produce medicine, therefore it is of high ornamental and economic value.In this research, we isolated the full length cDNA of Hydroperoxide lyase (HPL) from Daidai (Citrus aurantium ) petals by RT-PCR and RACE and tried to express this gene in E.coli. This laid a foundation for its application on subsequent genetic transformation study, which aims to control the expression of HPL gene and regulate he production of scents in plants, and provided a new approach to the genetic improvement of flower scents.1 Cloning of the full length cDNA of Hydroperoxide lyase by RT-PCR and RACEThe cDNA sequence of Daidai HPL is 1776 bp long. It contains an ORF encoding 499 amino acid residues (Mw=55.7 kDa), a 5' untranslated region of 52 bp and a 3' untranslated region of 224 bp. The protein encoded by this cDNA contains the four conserved regions of HPLs. We concluded that Daidai HPL is a hydrophilic protein. Compared with HPLs of other plants, Daidai HPL gene cDNA had 99%, 98% identity with that of sweet orange and rough lemon HPL genes, respectively. It had the lowest identity with that of cucumber and melon HPL genes, 33.3% and 33.2%, respectively. Mutialignment of the amino acid sequences showed that Daidai HPL and 13-HPLs of rough lemon, sweet orange, alfalfa, tomato, potato, pimiento, tobacco, guava and Arabidopsis belong to the same subgroup. This phylogenetic analysis suggested that Daidai HPL was a member of the 13-HPL family.2 Expression of Daidai HPL proteins in E. coll.The Daidai HPL gene cDNA was inserted into the vector pET-15b. The expression construct was transformed into BL21 and was induced. We found that the Daidai HPL proteins were expressed at high levels in E.coli. SDS-PAGE experiments showed that the Daidai HPL is 55 kDa. which is approximately the same with the calculated molecular weight 55.7kDa.3 Amplifition of Daidai HPL genomic fragmentGenomic fragment of Daidai HPL gene was also amplified by using PCR,and subsequent sequencing analysis showed that the Daidai HPL gene contained one intron of 2374bp. |
