烟草亚硝酸盐还原酶基因nii2的克隆及其功能分析
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摘要
亚硝酸还原酶(Nitrite Reductase, NiR)是一种可减少亚硝酸盐含量的酶,而亚硝酸盐是烟草特有亚硝胺(tobacco-specific nitrosamines, TSNA)的前体之一。烟草特有亚硝胺是潜在的致癌物质,只存在于烟草及烟草制品中。在硝酸盐代谢途径中,铁氧还蛋白-NiR催化亚硝酸盐还原成铵,因此调控NiR基因的表达会影响硝酸盐同化途径中亚硝酸盐的积累。迄今为止,多种高等植物的NiR cDNA已被克隆,但NiR基因的表达调控在不同的物种中不尽相同。本研究中,我们以烟草为材料,克隆了编码铁氧还蛋白-NiR nii2基因,并构建了植物表达载体pBI121-Ntnii2,以确定在烟草中过量表达该基因是否降低TSNA。此外,我们分析了转基因烟草和野生型烟草的Ntnii2基因表达情况,并检测了硝酸还原酶(Nitrate Reductase, NR)和NiR的活性,以及它们烟草外部过量的硝酸盐和烟草内部亚硝酸盐的反应。主要结果如下
1、克隆Ntnii2基因并构建植物表达载体：根据野生型烟草同源基因的信息,从烟草基因组中克隆Ntnii2基因,此基因全长1764bp,编码一条587个氨基酸的多肽。将Ntnii2连接到pBI121载体,构建植物表达载体pBI121-Ntnii2。
2、转基因植物的获得及分子鉴定：利用农杆菌介导法转化烟草,通过卡那霉素抗性筛选,得到23棵转基因烟草。利用RT-PCR检测,确定NtNii2基因能够被成功转录,并且且获得16株T1代种子,挑选其中8株做进一步的研究。RT-PCR结果表明转基因株系NiR基因的表达水平显著高于野生型。
3、转基因植物的生理特性分析：体内和体外NR活性研究中,选定的8株转基因株系的这两种酶活性均显著高于对应的野生型,在一些转基因株系中增加了约30%的活性。当硝酸盐含量减少近46%时,亚硝酸盐的含量在转化株和非转化株中差别不是十分明显。根据硝酸盐、亚硝酸盐和TSNA的研究,我们得出结论,转Ntnii2基因能够影响烟草的TSNA水平。
Nitrite Reductase (NiR) is an enzyme that can reduce the concentration of nitrite. Nitrite is one of the precursors of tobacco-specific nitrosamines (TSNA) which are potentially carcinogenic products and exist only in tobacco and tobacco products. In the second step of the nitrate assimilation pathway, ferredoxin-nitrite reductase (NiR) catalyzes the reduction of nitrite to ammonium, thus controlling this gene would influence the build-up of nitrite in nitrate assimilation pathway. In this study, we cloned the nii2gene from tobacco, and identified its function when it was overexpressed in tobacco. We further studied the relationship between nii2gene and TSNA, and investigated the NR and NiR activities, and their response to the excess amount of external nitrate and nitrite levels in tobacco. The detail results are summerised as follows:
1. Cloning of the Ntni2gene and construction of the plant expression vector, pBI121-Ntnii2
The Ntnii2gene was cloned from tobacco NC89based on the homologous information in Genbank (Accession number EST AB103507). The complete open reading frame (ORF) of Ntnii2gene contained1764bp in length andencoded587amino acids. The Ntnii2ORF was constructed into binary vector pBI121which has35S promoter of Cauliflower mosaic virus (CaMV), resulting in the pBI121-Ntnii2.
2. Transformation and characterization of Ntnii2gene
By using Agrobacterium-mediated transformation method,23transgenic tobacco lines were generated through kanamycin resistance screening. After RT-PCR-based screening, NtNii2gene was confirmed to be successfully transcripted and inherited into its16offsprings while8lines were selected for further generation. Semi-quantitative RT-PCR results showed that the NiR gene expression in transgenic lines was significantly higher than that in WT plants.
3. Study of physiological characters of transgenic plants
In vivo and in vitro NR activities were significantly higher in those selected8transgenic lines than the counterpart WT plants. Approximately30%NiR activity was increased in some transgenic lines. When the nitrate content showed nearly46%reduction, the nitrite contents were not considerably differed from that of non-transformed plants. As a consequence among nitrate, nitrite and TSNA, our results can conclude that transgenic action would influence TSNA levels in tobacco.
1、克隆Ntnii2基因并构建植物表达载体：根据野生型烟草同源基因的信息,从烟草基因组中克隆Ntnii2基因,此基因全长1764bp,编码一条587个氨基酸的多肽。将Ntnii2连接到pBI121载体,构建植物表达载体pBI121-Ntnii2。
2、转基因植物的获得及分子鉴定：利用农杆菌介导法转化烟草,通过卡那霉素抗性筛选,得到23棵转基因烟草。利用RT-PCR检测,确定NtNii2基因能够被成功转录,并且且获得16株T1代种子,挑选其中8株做进一步的研究。RT-PCR结果表明转基因株系NiR基因的表达水平显著高于野生型。
3、转基因植物的生理特性分析：体内和体外NR活性研究中,选定的8株转基因株系的这两种酶活性均显著高于对应的野生型,在一些转基因株系中增加了约30%的活性。当硝酸盐含量减少近46%时,亚硝酸盐的含量在转化株和非转化株中差别不是十分明显。根据硝酸盐、亚硝酸盐和TSNA的研究,我们得出结论,转Ntnii2基因能够影响烟草的TSNA水平。
Nitrite Reductase (NiR) is an enzyme that can reduce the concentration of nitrite. Nitrite is one of the precursors of tobacco-specific nitrosamines (TSNA) which are potentially carcinogenic products and exist only in tobacco and tobacco products. In the second step of the nitrate assimilation pathway, ferredoxin-nitrite reductase (NiR) catalyzes the reduction of nitrite to ammonium, thus controlling this gene would influence the build-up of nitrite in nitrate assimilation pathway. In this study, we cloned the nii2gene from tobacco, and identified its function when it was overexpressed in tobacco. We further studied the relationship between nii2gene and TSNA, and investigated the NR and NiR activities, and their response to the excess amount of external nitrate and nitrite levels in tobacco. The detail results are summerised as follows:
1. Cloning of the Ntni2gene and construction of the plant expression vector, pBI121-Ntnii2
The Ntnii2gene was cloned from tobacco NC89based on the homologous information in Genbank (Accession number EST AB103507). The complete open reading frame (ORF) of Ntnii2gene contained1764bp in length andencoded587amino acids. The Ntnii2ORF was constructed into binary vector pBI121which has35S promoter of Cauliflower mosaic virus (CaMV), resulting in the pBI121-Ntnii2.
2. Transformation and characterization of Ntnii2gene
By using Agrobacterium-mediated transformation method,23transgenic tobacco lines were generated through kanamycin resistance screening. After RT-PCR-based screening, NtNii2gene was confirmed to be successfully transcripted and inherited into its16offsprings while8lines were selected for further generation. Semi-quantitative RT-PCR results showed that the NiR gene expression in transgenic lines was significantly higher than that in WT plants.
3. Study of physiological characters of transgenic plants
In vivo and in vitro NR activities were significantly higher in those selected8transgenic lines than the counterpart WT plants. Approximately30%NiR activity was increased in some transgenic lines. When the nitrate content showed nearly46%reduction, the nitrite contents were not considerably differed from that of non-transformed plants. As a consequence among nitrate, nitrite and TSNA, our results can conclude that transgenic action would influence TSNA levels in tobacco.
引文
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