1.Molecular Cloning And Function Analysis Of UDPG Glucosyltransferase Gene 2.Molecular Cloning, Sequence Analysis And Evolution Of Actin And EF1α Genes In Stevia Rebaudiana

The pathway for steviol glycoside biosyntheses is shared with that of gibberellins. The highly activity nature and over expression of HMG-CoA reductase, CPS and KS would result in the production of a large amount of mevaloic acid, (-)-copalyl diphosphate and (-)-kaurene, the precursors of GAs and steviol in stevia rebaudiana leaves. To avoid the synthesis of an excess of GA, the precursor of which is also ent-kaurenoic acid must be converted to steviol, and then glucosylated into steviol glycosides, which accumulated in leaves at concentrations ranging from 10-30% of the leaf, dry weight.We report here the cloning and characterization of S. rebaudiana UDP-glucosyltransferase. Degenerate oligonucleotide primers were designed based on identical or highly conserved amino acid sequences of known flavonoid glucosyltransferase, and used to PCR amplify stevia glucosyltransferase gene fragments GTASE-I and GTASE-II. Two fragments with strong homology to known flavonoid glucosyltransferases were used to design other two primers to amplify 5' and 3'-half cDNA of stevia glucosyltransferase Gtl and Gt2, respectively. The full-length Gtl gene is 1568bp in length, with an open reading frame encoding 473 amino acids (Genbank?accession numbers AF515727). The full-length Gt2 gene is 1662bp in length, with an open reading frame encoding 454 amino acids (GenbankTM accession numbers AF548025). The predicted amino acid sequences of Gtl and Gt2 were 26-46% and 44-74% identical to the known UDPG glucosyltransferase respectively. The deduced amino acid sequences of Gtl and Gt2 were comfirmed to have one conserved region of glucosyltransferases, which is the region for the UDPG-glucose binding domain. Plasmid was prepared from recombinant clones and transformed into the E. coli BL21 DE3 strain for production of recombinant Gtl. The Gtl products of in vitro translation, which were ~55 kDa recombinant protein, hadanthocyanidins and steviol glucosyltransferase activity. Incubation of the purified recombinant Gtl with steviol and other aglycones in the presence of UDP-glucose donor substrate resulted in the formation of the glucosides. Relative active against a range of acceptor substrates, including steviol, cyanidin, delphinidin, peonidin and malvidin, was determined by HPLC analysis. Cyanidin gave the highest rates of glucosylation. The average rate of steviol glucosylation was approximately 33.5% toward that observed for cyanidin. Comparison the activity of the recombinant UDP-glucosyltransferase Gtl toward a range of acceptor substrates, suggests that it may participate in the synthesis of steviol glycosides. The results support the hypothesis that the flavonoid glucosyltransferases, which have broad substrate specificity, may be not only involving in flavonoid glucosylation but also playing a role to produce the water-soluble steviol-glycosides in S. rebaudiana.