A Study Of Transformation Of Nattokinase Into Lettuce

Nattokinase (NK, subtilisin NAT), which is produced by Bacillus subtilis var. natto, is a strong fibrinolytic enzyme in the traditional Japanese soybean food natto. The enzyme not only strongly hydrolyzes thrombi in vivo, but also converts plasminogen to plasmin.In the present study, 849bp gene fragment encoding NK was inserted into the pBI-121 vector, which is plant expression vector. The recombinent plamid, designated pBI-121-NK, was transformed into agrobacterium tumefaciens via the freeze and thaw method. The recombinent plamid, named LBA4404- pBI-121- NK, was selected to infect leaves of lettuce with the agrobacterium-medidated leaf disc transformation method. The result of transformation was assessed by PCR, RT-PCR analysis and sequence analysis, which indicated that NK gene had been integrated into genome of transgenic lettuce. This is the basic research for transgenic lettuce as bioreactor to large-scale produce nattokinase. Thereby, a series of cardiovascular diseases induced by thrombus could be prevented and cured conveniently through eating the raw transgenic lettuce.The results were as follows:1. The construction of the lettuce regeneration systemWe established two kinds of mediums: The inducing shoot medium is ( MS + 0.1mg/1 6-BA+0.05mg/l NAA), and the inducing root medium is (1/2MS+0.3mg/l NAA+0.5mg/l IBA+0.1% active carbon).2. The construction of pBI-121-NKPCR amplification of NK gene was carried out. After agarose gel electrophoresis, the amplified NK PCR product was purified and digested with Xba I and SacⅠ, ligated to the pBI-121 plasmid cut by the same restriction enzymes, introduced into Escherichia coli DH5α, the recombinent was named as the plant expression vector pBI-121-NK.3. The analysis of obtained transgenic resistant lettuce By transferring the above construct into agrobacterium tumefaciens strain LBA4404 using the freeze and thaw method, we gained a new vector and named LBA4404-pBI-121-NK.Lettuce was infected by using the agrobacterium - medidated leaf disc transformation.Transformants of lettuce were selected on kanamycin (50mg/l)-containing and carbenicillin (200mg/l)-containing mediums and propagated for subsequent analysis.4. The molecular analysis of the NK transgenic lettuceIn order to estimate the expression of NK in transgenic lettuce, callus or shoots of the transgenic lettuce were harvested from resistant mediums. Genomic DNA was isolated from the resistant lettuce, subsequently analysed by PCR and sequencing; Genomic RNA was extracted from the resistant lettuce, subsequently analysed by RT-PCR. The analysis revealed that NK gene was transformed into lettuce, which proved that the transgenic lettuce was obtained.