The Expression Of Thaumatin CDNA Gene In Tobacco

The sweet protein thaumatin was originally isolated from the fruit of a tropic plant Thaumatococcus daniellii Benth, which grows in forest areas of western Africa. It has advantageous properties of being nontoxic,low-caloric and noncariogenic.So it has the probability to take the place of sugar. However T.daniellifs growth demands a special phenological conditions. These harsh conditions make the natural source of thaumatin be so scarce.Therefore,as alternative to the production of thaumatin from its natural source,many attemps had been made to introduce and express thaumatin genes in recombinant hosts.Thaumatin genes were respectively cloned into Escherichia.coli, Bacillus subtilisjtreptomyces lividans and high plants under control of different promoters. The results, however, are disheartening. The recombinant thaumatin did not taste sweet.In this report,the two endonucleases EcoR I and EcoRV were used to digest the vector pBluescript SK,EcoRVand Pst I were used to digest pUC57,and the same time pUC18 was digested by EcoR I and Pst I .Then the three fragment were ligated into a recombinant vector-pUC18-tha.The thaumatin cDNA gene-tha were cloned into the expression vector pBI121 by use of vector pGEM7zf. Thaumatin cDNA was linked to one CaMV35S promoters and an express vector of thaumatin cDNA,pBI121-tha, was constructed. The pBI121-tha was transformed into agrobacterium LBA4404.The recombinant agrobacterium was introduced into tobacco.The genome DNA of tobacco was extracted from the transgenic tobacco.The PCR, PCR-Southern and Southern results confirmed that the tha gene was integrated into the tobacco genome.The total RNA of transgenic tobacco was extracted from the plant.The RT-PCR results revealed tha gene was transcripted into mRNA in the transgenic plant.The SDS-PAGE results and the sweet-testing,however,showed the thaumatin gene was not translated into the active protein.