Transformation Of Lactuca Sativa L. With GDP-D-Mannose Pyrophosphorylase And L-Galactose-1-Phosphatase Gene From Arabidopsis Thaliana L.

Vitamin C (L-ascorbic acid, AsA) is an essential micronutrient for human life. However, AsA can not be synthesized and stored in human bodies. AsA is obtained mainly from plants. In plants AsA is synthesized with D-mannose and L-galactose as key intermediates. GDP-D-mannose pyrophosphorylase (GDPMPPASE, EC2.7.7.22) catalyzes the synthesis of GDP-D-mannose and represents the first committed step in plant AsA biosynthesis. L-Galactose-1-phosphatase catalyzes the synthesis of L-Galactose. In order to improve the content of AsA in Lactuca sativa L., GDP-mannose pyrophosphorylase gene (gmp) and L-Galactose-1-phosphatase gene(galp)were cloned with specific primers according to the GenBank sequence from cDNA of Arabidopsis thaliana. CaMV 35S promoter, MYC sequence and NOS terminator were connected to gmp /galp gene respectively to constitute a gmp -containing expression cassette and a galp -containing expression cassette, the latter cassettes were transferred into pCAMBIA2301 expression vector respectively resulting in p2301-gmp-myc and p2301-galp-myc. L. sativa was transformed with Agrobacterium strain EHA105 containing p2301-gmp-myc or p2301-galp-myc through Agrobacterium-mediated leaf-disc transformation, and 64 gmp transgenic plants and 56 galp transgenic plants were obtained. PCR, Southern Blot and Real-Time PCR confirmed the integration and expression of the introduced gmp /galp in transgenic L. sativa plants. The contents of AsA in transgenic L. sativa plants were measured by HPLC–ELSD. The results indicated that most of the transgenic plants had higher AsA contents than those of wild type plants, with the highest content of AsA being about 2.5 times(gmp) and 1.9 times(galp) of the wild type. This study demonstrates that over-expressing the gmp /galp gene is an effective method to enhance AsA content in L. sativa L.