Studies On The Introduction Of Nattokinase Gene In Lettuce (Lactuca Sativa)

In this experiment, apotential thrombolytic medicine Nattokinase gene was introduced into lettuce(Lactuca sativa) with Agrobacterium transformation system and biolistic method. The experiment results covered several following points:(1)DNA fragment encoding nattokinase was amplified by PCR from the genome DNA of Bacillus natto , and cloned into PUCm-T vector, then sequenced, which was the same as Genebank' s Nattokinase.(2)NK was cloned into nuclear plasmid PBG, PB and chloroplast expression plasmid PLCTB . The expression vectors of fusing nk, namely PBG-NK, PB-NK, PLCTB-NK were constructed by gene recombination technology.(3)To differ the transgenic lettuces from the whole materials, suitable concentration of kanamycin was 100mg/L and spectinomycin was 20mg/L.(4)Lettuces were infected with Agrobacterium tumefaciens Comn LBA4404 containing Nattokinase gene, under the control of 35S promoter of cauliflower mosaic virus or PI II promoter of potato proteinase inhibitor II, the former plasmid named PBG-NK and the later called PB-NK. The factors influencing Agrobacterium mediated lettuce transformation were studied. The optimal duration of pre-culture was two days, The suitable infection duration was five minutes and the appropriate co-culture duration was two days.(5) And the sturdy regenerated plants with kanamynic resistant were obtained. PCR and PCR-Southern blot analysis of lettuce DNA confirmed that nk gene had been integrated into the plant genome. The results also showed that the transform frequency of PBG-NK was higher than that of PB-NK.(6) The chloroplast expression plasmid PLCTB-NK which contained lettuce psb promoter and terminor, besides two homogeneous sequences at nk gene flanks was transformed to lettuces by biolislic method. Because selective cycle of chloroplast transformation was longer than the nuclear transformation and the research time was limited, this experiment only obtained the weakly regenerated plants with spectinomymc resistant and further molecular detections should go on.