Cloning And Genetic Transformation Of The Key Gene(UGT76G1) In The Steviol Glycoside Biosvnthesis

Steviol glycoside is a new natural sweetener extracted from the leaves of Stevia rebaudiana which belongs to composite plants.It has unique properties of high sweetness and low calorie.UGT76G1(GenBank NO:AY345974)gene is cloned in stevia.UGT76G1 gene is related to the metabolism of SGs.It was confirmed that the St were converted to RA by UGT76G1.Overexpressing of this gene may increase the content of RA.In this paper,the UGT76G1 gene was cloned from stevia 'shoutian-2',and the plant overexpressing expression vector were constructed,then they were introduced into stevia by Agrobacterium-mediated transformation.The main results are followed:1.The UGT76G1 gene overexpressing expression vector was constructed,The correct insertion of the exogenous gene in the vectors was confirmed by PCR,enzyme and sequencing.2.Establishment of efficient transformation and regeneration system from shoot tips of stevia 'shoutian-2'.The results showed that the optimum genetic transformation system could be achieved with minute infection of Agrobacterium rhizogenes A4(OD600 = 0.6,100?mol/L AS)and 3 days co-culture.3.Genetic transformation of 'shouyian-2' mediated by Agrobacterium rhizogenes A4.The kan-resistant plantlets were obtained after the shoot tips of'Jinshoutian-2' infected by A4 with plant expression vector pRI 101 AN-DNA.5 kan-resistant plants were obtained after the infection of A4 with pRI 101 AN-DNA:::UGT76G1 were confirmed by PCR,which preliminarily verified the overexpressing gene had been integrated into the genome.