体外改组强耐盐基因NHXFS1在烟草中的表达及耐盐性分析  

Expression and Salt Tolerance Analysis of a Novel Na^+/H^+ Antiporter Gene NHXFS1 in Tobacco (Nicotiana benthamiana L.)

作　　者：黄继斌[1] 张辉[1] 郭力榕[1] 徐源[1] 夏涛[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062

出　　处：《农业生物技术学报》2012年第7期774-781,共8页Journal of Agricultural Biotechnology

基　　金：上海市教委创新基金(No.11ZZ40)

摘　　要：位于植物液泡膜的 Na+/H+逆向转运蛋白(NHX1)是将 Na+区隔化到液泡中的主要蛋白,对植物耐盐性起着极其重要的作用。NHXFS1 基因是以拟南芥(Arabidopsis thaliana)、水稻(Oryza sativa)和菊花(Flos chrysanthemi)的液泡膜 Na+/H+逆向转运蛋白基因 AtNHX1、OsNHX1 和 DmNHX1 为模板,通过 DNA家族改组技术,对其进行体外定向分子进化获得的一个功能显著增强的新基因。本研究利用根癌农杆菌(Agrobacterium tumerfaciens)介导的叶盘法将 NHXFS1 基因转入烟草 (Nicotiana benthamiana)中,通过潮霉素抗性筛选出再生植株,经PCR 和 Southern blot 鉴定证实 NHXFS1 基因成功地整合到烟草基因组中。耐盐性测试结果显示,高盐胁迫条件下,萌发期和幼苗期的转基因烟草长势明显优于对照组,与对照组相比,转基因烟草叶片中积累了更多的脯氨酸和Na+。 RT-PCR和 Real-time PCR 结果表明,NHXFS1 基因在烟草中正常表达,盐胁迫条件下NHXFS1基因在根、茎、叶中的表达量分别上调了 1.01、0.60 和 1.79倍,跟对照组相比有明显提高(P<0.05)。研究结果提示,体外改组获得的新基因 NHXFS1 在植物耐盐基因工程和分子育种中具有较大的应用价值,同时也证明通过基因改组方法开发新基因用于改良植物耐盐性是可行的。Plant tonoplast Na＋/H＋ antiporter comparting sodium into vacuolar plays an important role in salinity tolerance. NHXFS1 is a novel tonoplast Na＋/H＋ antiporter gene which is obtained from AtNHX1 of Arabidopsis thalio, OsNHX1 of Oryza sativa and DmNHX1 of Flos chrysanthemi by DNA family shuffling in vitro. To study the expression pattern and salt tolerance ability of NHXFS1 in plant, we transformed NHXFS1 into tobacco （Nicotiana b enthamiana） by Agrobacterium-mediated leaf disc method, the transgenic plants were screened with hygromycin B and identified by PCR and Southern blot. 15 NHXFS1 positive plants were identified from 43 hygromycin-resistant tobacco plants that we had gotten. Southern blotting analysis with NHXFS1 probe indicated that the NHXFS1 gene was integrated into tobacco genome. According to segregate ratio, transgenic tobacco line with single copy was inbreeded to homozygote. The result of salinity tolerance test demonstrated that the growth vigor of transgenic plants under high salt stress was better than that of the control ones. The transgenic seeds could still germinate in MS plate with 400 mmol/L NaC1, but the controls could not. Also, the growth statuses of transgenic plants were better than that of controls in soil contained high NaCI concentration. When NaC1 concentration reached 300 mmol/L, the wild-type plants were nearly wilting, while the transgenic ones were still vital. Results showed that the transgenic plants could accumulate more proline and Na＋ ion. RT-PCR analysis showed that the NHXFS1 gene was expressed in tobacco. The result of Real-time PCR showed that the transcript level of NHXFS1 was significantly higher in different tissues after 200 mmol/L NaC1 treatment, which increased 1.01, 0.60 and 1.79 fold in root, stem and leaf, respectively. The data suggest that this novel gene NHXFS1 can be applied in plant salt tolerance engineering and molecular breeding. And it also manifest that it＇s available to develop novel genes improving plant salt tolerance by DNA family

关 键 词：NHXFS1基因 烟草 NA+/H+逆向转运蛋白 耐盐性 DNA家族改组 

分 类 号：S572[农业科学—烟草工业]