High Expression Of Codon Optimized And Synthesized Nattokinase Gene In Melon And Tomato Fruits

Nattokinase(NK, Subtilisin NAT), an alkaline serine protease produced by Bacillus subtilis var. natto, showed high fibrinolytic activity by direct and indirect thrombolytic ways. NK is safe, oral administration, convenient, low-cost and efficacious, these advantages made defined NK as a novel oral thrombolytic agent to prevent and treat cardiovascular diseases.In this paper, the aprN(eNK) gene was cloned from Bacillus subtilis var. natto. A synthetic gene (sNK) encoding NK was constructed by modifying its nucleotide sequence based on the optimized codon usage in plants, but the amino acid sequence of the mature NK was not altered. eNKi and sNKi gene incorporating the first intron from the tomato E8 gene were also constructed by overlap extension PCR method. The plant binary expression vector pPZP221(35S-nos) was used as the initial vector, the four nattokinase gene——eNK, eNKi, sNK and sNKi, which were inserted Kozak sequences before ATG codon, were cloned into the downstram of constitutive CaMV 35S promoter or fruit specific E8 promoter. The 8 plant expression vectors: p35S-eNK, pE8-eNK, p35S-eNKi, pE8-eNKi, p35S-sNK, pE8-sNK, p35S-sNKi and pE8-sNKi were constructed and transformed into Agrobacterium tumefaciens LBA4404 by the freeze-thaw method.The four nattokinase gene were transiently expressed in melon(Cucumis melo L. cv Hetao) fruit by agroinfiltration. Orthogonal tests in melon fruit determined the optimal agroinfiltration as:an acetosyringone concentration of 250 μmol/L, an OD6oo=0.6 and harvesting 5 days after infiltration. The expression level of four genes in tomato fruit were measured by real-time quantitative reverse transcription-PCR (RT-qPCR) analysis, and fibrinolytic activity were detected through fibrin plate method. RT-qPCR results showed that the expression levels of sNK and sNKi gene were siginificantly higher than that of the non modified eNK and eNKi gene. The average expression of sNK gene under the control of 35S promoter was 8.64 times higher than that of eNK gene, and the average expression of sNKi gene under the control of E8 promoter was 10.93 times of eNKi gene. The expression level of recombinant NK controlled by fruit-specific E8 promoter was significantly higher than that controlled by constitutive 35S promoter, and the highest expression level appeared in mid-mature of melon fruit. Intron can be correctly splicing in the post transcription process of sNKi gene. The expression level of sNKi gene was higher than that of sNK gene which without intron though their transcription products were the same. Those proved that intron can significantly improve the transient expression of sNK gene. The results of fibrinolytic activity were consistent with the results of RT-qPCR. Fibrinolytic activity was detected in transiently expressed samples of two synthetic genes, which indicated that the target genes can be normally translated and show thrombolytic activity in melon fruits. The highest fibrinolytic activity of sNKi gene controlled by E8 promoter was 237.90 U/g in melon fruits.Transient expression of sNK and sNKi gene in Moneymaker and Micro-Tom tomato(Lycopersicon esculentum Mill) fruits was similer to melon fruits. The expression of two synthetic nattokinase genes can be detected in tomato at veraison, maturation and full ripeness stage, but not at breaker stage. The highest fibrinolytic activity of sNKi gene can reach to 144.27 U/g under the control of E8 promoter.Two synthetic nattokinase genes were stably transformed into three tomatos— Moneymaker, M82 and Micro-Tom by agrobacterium infection. There were 32 strains of p35S-sNK To transgenic tomato plants,27 strains of pE8-sNK To plants,21 strains of p35S-sNKi To plants and 27 strains of pE8-sNKi plants by PCR analysis, which indicated that the target gene had been integrated into tomato genome. T1 transgenic tomato plants were obtained after generation-adding cultivation.11 strains of p35S-sNK transgenic tomato plants were detected transcriptional expression and 7 strains showed fibrinolytic activity; 12 strains of pE8-sNK transgenic tomato plants were detected transcriptional expression and 7 strains showed fibrinolytic activity; 8 strains of p35S-sNKi transgenic tomato plants were detected transcriptional expression and 6 strains showed fibrinolytic activity; 11 strains of p35S-sNK transgenic tomato plants were detected transcriptional expression and 10 strains showed fibrinolytic activity. The maximum fibrinolytic activity of Moneymaker strain AD 1-3 was 30.69 U/g. The T1 transgenic tomato plants which showed high fibrinolytic activity continued to be cultured, and T2 transgenic tomato plants were obtained.9 strains 28 plants of p35S-sNK transgenic tomato,8 strains 27 plants of pE8-sNK transgenic tomato,8 strains 25 plants of p35S-sNKi transgenic tomato and 10 strains 36 plants of pE8-sNKi transgenic tomato were detected the expression of nattokinase gene. The expression of fibrinolytic activity in Moneymaker AD 1-3-3 plant was up to 63.90 U/g.