人工合成的纳豆激酶基因转化烟草的研究

生物技术通报 ›› 2014, Vol. 0 ›› Issue (10): 94-100.

人工合成的纳豆激酶基因转化烟草的研究

刘靓蕾, 张妍, 孙晓蕾, 韩岚, 满达呼斯琴, 哈斯阿古拉

内蒙古大学生命科学学院内蒙古自治区牧草与特色作物生物技术重点实验室,呼和浩特 010021

收稿日期:2014-03-03 出版日期:2014-10-20 发布日期:2014-10-17
作者简介:刘靓蕾,女,硕士,研究方向：植物分子生物学及基因工程
基金资助:
内蒙古自治区“草原英才”工程资助项目（内组通字[2013]17号）

Research of Transferred Synthetic Nattokinase Gene in Tobacco Genome

Liu Jinglei, Zhang Yan, Sun Xiaolei, Han Lan, Kh.Mandakhtsetsen, Hasi Agula

Inner Mongolia Key Laboratory of Herbage ＆ Endemic Crop Biotechnology,College of Life Sciences,Inner Mongolia University,Huhhot 010021

Received:2014-03-03 Published:2014-10-20 Online:2014-10-17

摘要/Abstract

摘要： 根据植物偏爱密码子人工合成的纳豆激酶基因sNK和其中插入番茄果实特异表达基因E8第一内含子的纳豆激酶基因sNK-E8i,通过农杆菌侵染的方法转化到烟草NC89中。经PCR检测,得到12株转sNK基因烟草植株和10株转sNK-E8i基因烟草植株,初步证明目的基因已整合到烟草基因组中; RT-PCR检测有9株转sNK基因烟草植株和10株转sNK-E8i基因烟草植株为阳性; 通过RT-qPCR将两种基因在烟草中的表达量进行比较,结果表明转sNK-E8i基因的植株中纳豆激酶的表达量比转sNK基因的植株中高; 通过纤维蛋白平板法检测其活性,共有5株转sNK基因烟草植株和3株转sNK-E8i基因烟草植株检测到溶圈,说明目的基因在部分转基因烟草中可正常转录和翻译并表现出溶栓活性。经加代培养已得到T代转基因植株。

关键词: 纳豆激酶, 人工合成, 植物偏爱密码子, 烟草, 纤维蛋白平板法

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

刘靓蕾, 张妍, 孙晓蕾, 韩岚, 满达呼斯琴, 哈斯阿古拉. 人工合成的纳豆激酶基因转化烟草的研究[J]. 生物技术通报, 2014, 0(10): 94-100.

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.

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