Expression Of Earthworm Fibrinolytic Enzyme Encoding Gene In Tomato Fruit

Earthworm Fibrinolytic Enzymes belong to serine protease family, found in midgut of earthworm. They can both elicit lysis of existing thrombus and inhibition of new thrombus formation. Drugs containing EFE has been adopted clinically for years in China. In this assay, we used EFE 7# encoding gene as the target gene for genetic transformation of cherry tomato, which will be useful in creation of novel tomato variety with thrombus formation inhibitory activity.First, the recombinant plasmids were introduced into agrobacterium EHA105 by tri-parental conjugation. Gene of interest was then introduced into tomato plants via high efficient agrobacterium-mediated transformation. Finally, the regenerated plants were obtained and its molecular and biochemical identification were carried out.The results showed that the tri-parental conjugation is very efficient method for transformation of agrobacteria. Tissue culture medium was experimented. The OD600 of optimal concentration of agrobacterium is 0.3. Suitable infection time is 10min. Thus, a set of efficient and simple agrobacterium-mediated transformation system for tomato was established. Genomic DNA of regenerated plants was extracted, and 22 transgenic plants obtained were screened out as positive plants after the preliminary identification with PCR. Casein plate experiments showed that several transgenic plants had a certain degree of earthworm fibrinolytic enzyme activity. Semi-quantitative RT-PCR analysis failed to detect EFE mRNA transcript due to bad RNA quality.