The Expression Of Thaumatin CDNA In Maize And The Co-expression Of Thaumatin And PDI In Tobacco

Thaumatin, 'the sweetest substance known', is a protein produced by the plant Thaumatococcus daniellii Benth. It is an intensely sweet protein (100,000 times sweeter than sucrose on a molar basis), which has been approved for human consumption, and is currently used in products such as chewing gum, dairy produce and Pharmaceuticals because of its low calories, high sweetness and innocuity. However, Thaumatococcus daniellii Benth can't seed outside its producing area. But gene engineering can solve this problem.Maize is a main crop. If sweet enough, the maize can promote the livestock eating and weighting. So I wish, through the gene engineering technique, the thaumatin can express in the maize. Meanwhile, the system, the high efficiency transformation of maize mediated by Agrobacterium tumefaciens, will be further improved.After successfully constructing a new botanic expressing vector pCAMBIA1302-tha containing the sweet-protein thaumatin cDNA gene, the recombinant plasmid was transformed into the calli of maize R18 via agrobacterium LBA4404. Through the organ culture technology, we have gained the regenerating transgenic plants. The genome DNA of transformed maize's leaves was extracted. It proved that thaumatin cDNA gene has integrated into the genome of transformed maize plants by PCR, PCR-Southern and Southern blotting analysis. Extracting the whole RNA from the leaves of transgenic plants, and the RT-PCR and Northern blotting results confirmed that the thaumatin cDNA gene was transcripted into mRNA. Isolating the all dissolvable proteins from the leaves of these plants. The result of SDS-PAGE showed the same molecular weight protein as Thaumatin in the transgenic maize, not in untransgenic maize. On the whole, it illuminates the thaumatin cDNA gene has translated into the protein, only we can't confirm it is Thaumatin. At present, because we haven't gained the antibody of Thaumatin, it cannot be proved that it is Thaumatin by the way of Western blotting analysis. At the same time, the leaves of these transgenic maize were not sweet. All of the words, the thaumatin cDNA gene has not expressed the active protein in the leaf cells of transgenic maize.We have not gained the sweet maize. Of course, it is due to a lot of reasons. For example, the fringes of transgenic maize (The Thaumatin is produced by the plant Thaumatococcus daniellii Benth in the gelatinous arils of the fruit, so we expect it will correctly express in the fringes of transgenic maize), which have not maturated, have not done the sweet analysis. So it cannot confirm the transgenic maize unsweet. But we form the transformation system of maize mediated by Agrobacterium tumefaciens.