Research of Transferred Synthetic Nattokinase Gene in Tobacco Genome

Abstract

Abstract: The nattokinase gene sNK which was artificially synthesized according to plant preferential codons, and the nattokinase gene sNK-E8i which has been got by inserting the first intron of tomato specifically expressed gene E8 into sNK, were transformed into tobacco NC89 through Agrobacterium-mediated method. 12 strains of sNK transgenic tobacco plants and 10 strains of sNK-E8i transgenic tobacco plants were acquired through PCR test. It preliminarily demonstrated that the target genes were integrated into tobacco genomes. It was found that 9 strains of sNK transgenic tobacco plants and 10 strains of sNK-E8i transgenic tobacco plants were positive by using RT-PCR. The result showed that the expression level of sNK-E8i transgenic tobacco plants was higher compared with that of sNK transgenic tobacco plants by RT-qPCR. Besides, the enzyme activity was tested through agarose-fibrin plate analysis, and the dissolving circles of 5 strains of sNK transgenic tobacco plants and 3 strains of sNK-E8i transgenic tobacco plants were detected. The result showed that target genes could be transcribed and translated normally in a part of transgenic tobacco and presented the activity of thrombolysis. The T₁ transgenic straints were obtained by generation-adding cultivation.

Key words: Nattokinase, Synthetic, Plant preferential codon, Tobacco, Agarose-fibrin plate analysis

Liu Jinglei, Zhang Yan, Sun Xiaolei, Han Lan, Kh.Mandakhtsetsen, Hasi Agula. Research of Transferred Synthetic Nattokinase Gene in Tobacco Genome[J]. Biotechnology Bulletin, 2014, 0(10): 94-100.

References

[1] H, Hamada H, Tsushima H, et al. A novel fibrinolytic enzyme（nattokinase）in the vegetable cheese Natto;a typical and popular soybean food in the Japanese diet[J]. Experientia, 1987, 43（10）：1110-1111.
[2] T, Yamagata Y, Ichishima E. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis（natto）[J]. Bioscience, Biotechnology, and Biochemistry, 1992, 56（11）：1869-1871.
[3] 李晶, 王玉霞, 等.纳豆激酶基因的表达及纯化[J].生物技术, 2004, 13（6）：12-15.
[4] 谢玉娟, 李申, 等.纳豆激酶基因的克隆及其在大肠杆菌和枯草芽孢杆菌中的表达[J].食品科学, 2007, 28（5）：199-202.
[5] 徐祥, 王婷婷, 等.纳豆激酶基因在毕赤酵母中的表达纯化及抗体制备[J].中国生化药物杂志, 2010（1）：10-13.
[6] 杨凌：西北农林科技大学, 2005.
[7] 2009.
[8] 范三红, 郭蔼光, 单丽伟, 等.拟南芥基因密码子偏爱性分析[J].生物化学与生物物理进展, 2003, 30（2）：221-225.
[9] 李瑞芳.同义密码子的使用偏好性对蛋白质折叠速率的影响[J].生物物理学报, 2013, 29（8）：603-613.
[10] X. How optimized is the translational machinery in Escherichia coli, Salmonella typhimurium and Saccharomyces cerevisiae?[J]. Genetics, 1998, 149（1）：37-44.
[11] T, Müllertz S. The fibrin plate method for estimating fibrinolytic activity[J]. Archives of Biochemistry and Biophysics, 1952, 40（2）：346-351.
[12] 高政权.钝顶螺旋藻藻蓝蛋白提取和纯化工艺研究进展[J].食品研究与开发, 2007, 28（9）：151-154.
[13] A, Sommergruber K, Thompson D, et al. The expression of a nopaline synthase—human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue[J]. Plant Molecular Biology, 1986, 6（5）：347-357.
[14] E, Christou P, Gianì S, et al. Introns are key regulatory elements of rice tubulin expression[J]. Planta, 2004, 218（5）：693-703.
[15] R, Bailey-Serres J. Regulation of translational initiation in plants[J]. Current Opinion in Plant Biology, 2002, 5（5）：460-465.
[16] C, Laufs J, Grant S, et al. The combination of a novel stimulatory element in the first exon of the maize Shrunken-1 gene with the following intron 1 enhances reporter gene expression up to 1000-fold[J]. Plant Molecular Biology, 1991, 16（2）：199-207.
[17] JH, Lee SY, Yu HJ, et al. Petunia actin-depolymerizing factor is mainly accumulated in vascular tissue and its gene expression is enhanced by the first intron[J]. Gene, 2002, 292（1）：233-243.
[18] YM, Mun JH, Kim H, et al. An upstream region in the first intron of petunia actin-depolymerizing factor 1 affects tissue‐specific expression in transgenic Arabidopsis（Arabidopsis thaliana）[J]. The Plant Journal, 2007, 50（2）：230-239.
[19] Sousa SM, del Giúdice Paniago M, Arruda P, et al. Sugar levels modulate sorbitol dehydrogenase expression in maize[J]. Plant Molecular Biology, 2008, 68（3）：203-213.
[20] 葛鸿飞.在转基因水稻植株中蜡质基因第1内含子对基因表达影响的分析[J].植物生理学报, 2001, 27（5）：381-386.