Hu Lei, Lu Hai, Liu Qunlu, Chen Xuemei, Jiang Xiangning
The Tree and Ornamental Plant Breeding and Biotechnology Laboratory of State Forestry Administration, Beijing 100083, P.R. China.
Tree Physiol. 2005 Oct;25(10):1273-81. doi: 10.1093/treephys/25.10.1273.
The mtlD gene encoding mannitol-1-phosphate dehydrogenase, which catalyzes the biosynthesis of mannitol from fructose, was cloned from Escherichia coli and transferred to poplar (Populus tomentosa Carr.) through Agrobacterium-mediated transformation. The transgenic plants were screened and selected on Murashige and Skoog (MS) medium containing 30-50 mg l(-1) kanamycin and verified by polymerase chain reaction (PCR) and Southern blotting. Expression of the gene led to synthesis and accumulation of mannitol in the transgenic plants. Gas chromatography and mass spectrometry (GC/MS) and capillary gas chromatography (GC) showed that transgenic plants accumulated much more mannitol in their tissues than the wild-type plants, whether cultured in vitro, or grown hydroponically or in the field. Increased salt tolerance of transgenic plants was observed both in vitro and in hydroponic culture. The transgenic buds rooted normally on MS medium containing 50 mM NaCl, whereas wild-type buds did not. In the 40-day hydroponic experiments, transgenic poplar plants survived in a 75-mM NaCl treatment, whereas the wild-type poplar plants tolerated only 25 mM NaCl. Under the same NaCl stress, stomatal conductance, transpiration rates and photosynthetic rates were all higher in transgenic plants than in wild-type plants, whereas cellular relative conductivity was lower. We demonstrated that the mtlD gene was expressed in transgenic poplar plants, resulting either directly or indirectly in mannitol accumulation and improved salt tolerance. The constant mannitol concentrations in transgenic plants during the NaCl treatments indicated that mannitol accumulation caused by the mtlD gene was not a consequence of NaCl stress. Height growth was reduced by about 50% in the transgenic plants compared with the wild-type plants in the absence of salt; however, relative growth rate was much less influenced by salt stress in transgenic plants than in wild-type plants. The stunted growth of the transgenic plants may in part explain their improved salt tolerance.
编码催化从果糖生物合成甘露醇的甘露醇-1-磷酸脱氢酶的mtlD基因，从大肠杆菌中克隆出来，并通过农杆菌介导的转化转移到杨树（毛白杨）中。转基因植物在含有30-50 mg l(-1)卡那霉素的Murashige和Skoog（MS）培养基上进行筛选和选择，并通过聚合酶链反应（PCR）和Southern印迹进行验证。该基因的表达导致转基因植物中甘露醇的合成和积累。气相色谱-质谱联用（GC/MS）和毛细管气相色谱（GC）表明，无论是在体外培养、水培还是田间生长，转基因植物组织中积累的甘露醇都比野生型植物多得多。在体外和水培中都观察到转基因植物的耐盐性增强。转基因芽在含有50 mM NaCl的MS培养基上能正常生根，而野生型芽则不能。在为期40天的水培实验中，转基因杨树植株在75 mM NaCl处理下存活，而野生型杨树植株仅能耐受25 mM NaCl。在相同的NaCl胁迫下，转基因植物的气孔导度、蒸腾速率和光合速率均高于野生型植物，而细胞相对电导率较低。我们证明mtlD基因在转基因杨树植株中表达，直接或间接地导致甘露醇积累和耐盐性提高。在NaCl处理期间，转基因植物中甘露醇浓度恒定，这表明由mtlD基因引起的甘露醇积累不是NaCl胁迫的结果。在无盐条件下，转基因植物的高度生长比野生型植物降低了约50%；然而，转基因植物的相对生长速率受盐胁迫的影响比野生型植物小得多。转基因植物生长发育迟缓可能部分解释了它们耐盐性的提高。
