Construction Of Plant Expression Vector On Nattokinase And Establishing Of Tomato Genetic Transformation System

Tomato is one of the important vegetables and crops,which has the largest planting area and the highest annual output in the world. At the same time, tomato is an important object of stuady and model plant in subjects study of genetics and bioengineering, whose researching achievements are usually used for reference in other crops. Till to the end of 1997,48 kinds of transgenic tomatos had already produced commercially. At present, tomato is acted as bio-reactor in order to manufacture plant vaccine,gene engineer pharmaceutical and function food which is becoming a hot question in the study of tomato gene engineer. Nattokinase, which was extracted from the Japanese traditional food-natto, is derived from the Bacillus natto and found to be a strong fibrinolytic serine enzyme. The results at home an abroad indicate,nattokinase can effectivlly decompose the main component of thrombus-fibrin, and it is developed a new kind of the ideal thrombolytics pharmaceuticals in large possibility. Therefore, the aim of this study is to establish a high-efficient genetic transformation system of tomato mediated by Agrobacterium tumefaciens in order that a new approach is provided by using other plants as bio-reactors to obtain nattokinase protein. Now, these are the study achievements : 1. According to the NK DNA sequence published in GeneBank database, we design the NK premer and add to BamHI engzme points on the both sides, then a PCR reaction is proceeded in by liquid Bacillus natto . NK gene is extracted and purified by way of the recycled,reagent kit,at the same time, NK is ligased to the vector PBST, thus NK gene is successfully cloned. The test results of DNA sequence indicated that this gene is 1143 bp , with encoding 381 amino acids.The homology with reported gene(AF368283)in GeneBank database was 99.7% ,however, the three abrupt bases did not influence the fibrinolytic activity of nattokinase protein . 2. By the plismid pBPC47 ,pCAMBIA1300 and pMG15,we constructed the expression vector of NK gene.The test results of PCR reaction and enzyme identification proved target gene was positively inserted into the plasmid pCAMBIA1300 ,thus the expression vector of NK gene was successfully constructed. 3. The expression vector was transferred into Agrobacterium tumefaciens LBA4404 using the freeze-thaw method, then the plasmid pCAMBIA-nk was introduced into tomato by Agrobacterium-mediated transferring. Many resistant callus were obtained through selection of Hptromycin, at last,7 regenrated tomatoes were available,and the maximal transformation rate was 7.52‰. 4. With the plasmid pCAMBIA-nk as positive control and untransplanted tomato as negative control , the PCR analysic result of 7 regenerated pants showed that 7 regenerated pants of them had amplified 1146bp DNA band ,which had the same location as amplification band of the positive plasmid . these proved that foreign NK gene have been integrated into tomato genome.