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为研究荷花液泡膜型Na+/H+逆向转运蛋白基因NnNHX1的耐盐性,构建了植物表达载体pCAMBIA1301-220-NnNHX1,并通过农杆菌介导的叶盘转化法将NnNHX1基因转入烟草。利用荧光定量PCR分析NnNHX1在转基因烟草中的表达情况。对转基因烟草进行盐胁迫试验,并测定其在盐胁迫条件下叶绿素、脯氨酸和丙二醛含量、相对电导率、SOD和POD活性等相关生理指标及后代种子的发芽率。结果显示,在获得的7个转NnNHX1基因烟草株系中,T-N1、T-N2和T-N10等3个株系NnNHX1表达量较高。盐胁迫下,转基因烟草组培苗和盆栽苗的生长状况都好于对照植株,在叶绿素含量、脯氨酸含量、细胞质膜的完整性(相对电导率和丙二醛含量)和抗氧化酶(SOD和POD)活性等方面都好于对照,并且后代种子的发芽率也明显高于对照。表明荷花NnNHX1基因能够在烟草中正常翻译和表达,NnNHX1的过量表达提高了转基因烟草的耐盐性。
In order to study the salt tolerance of the vacuolar membrane type Na + / H + antiporter gene NnNHX1, a plant expression vector pCAMBIA1301-220-NnNHX1 was constructed. The NnNHX1 gene was transferred into tobacco by Agrobacterium tumefaciens-mediated leaf disc transformation. The expression of NnNHX1 in transgenic tobacco was analyzed by fluorescence quantitative PCR. The transgenic tobacco plants were subjected to salt stress and the contents of chlorophyll, proline and malondialdehyde (MDA), relative conductivity, activities of SOD and POD and their germination rate under salt stress were measured. The results showed that NnNHX1 was highly expressed in three lines of TnN1, T-N2 and T-N10 among the 7 transgenic lines of NnNHX1. Under salt stress, the growth of transgenic tobacco plantlets and potted seedlings were better than the control plants. Under the conditions of chlorophyll content, proline content, plasma membrane integrity (relative conductivity and malondialdehyde content) and antioxidant enzymes ( SOD and POD) activities were better than the control, and the germination rate of progeny seeds were significantly higher than the control. The results showed that the NnNHX1 gene could be normally translated and expressed in tobacco and the overexpression of NnNHX1 increased the salt tolerance of transgenic tobacco.