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为获得抗旱和耐盐性提高的甘蓝植株,通过农杆菌介导法将来自菠菜的甜菜碱醛脱氢酶(Betaine Aldehyde Dehydrogenase,BADH)基因导入甘蓝品系03079,并采用正交设计优化影响转化效率的参数,建立了甘蓝高效转化体系,即以侵染液为AA液体培养基、乙酰丁香酮200μmolL-1、侵染时间20min、共培养天数2d为最佳转化参数,在该条件下转化率可达54.26%。转基因甘蓝植株经PCR检测初步说明BADH基因已导入甘蓝中,Southern杂交证明BADH基因已稳定整合到甘蓝基因组中。甜菜碱脱氢酶活性测定结果表明,经过聚乙二醇(PEG)、NaCl和干旱处理的转基因甘蓝植株的BADH酶的平均比活力范围在2.1Umg-1～3.6Umg-1之间,不同处理的转基因株系酶比活力显著高于相应的未转基因株系。膜的相对电导率测定结果说明,经过PEG、NaCl和干旱处理的转基因植株平均相对电导率在16.2%～32.6%之间,耐逆境胁迫处理后的绝大多数转基因株系相对电导率显著低于相应对照。多数转BADH基因甘蓝植株在干旱、盐胁迫和PEG胁迫条件下生长势强于未转基因植株,表现为大多数转基因株系株高增幅显著高于对照,说明BADH基因的导入能提高转基因甘蓝植株的抗旱和耐盐性。我们获得的抗旱和耐盐能力明显提高的转基因甘蓝植株,可作为培育耐盐、抗旱甘蓝品种的种质材料。
To obtain the cabbage plants with increased drought and salt tolerance, the Betaine Aldehyde Dehydrogenase (BADH) gene from spinach was introduced into Brassica napus line 03079 by Agrobacterium tumefaciens-mediated method. The orthogonal design was used to optimize the transformation efficiency , The optimal transformation parameters were established for the efficient transformation system of cabbage. The optimal transformation parameters were as follows: inoculum liquid AA liquid medium, acetosyringone 200 μmol L -1, infection time 20 min, co-culture days 2d, Up to 54.26%. The results of PCR showed that BADH gene had been introduced into cabbage and Southern hybridization proved that BADH gene was stably integrated into cabbage genome. The result of betaine dehydrogenase activity showed that the average specific activity of BADH enzyme in transgenic cabbage plants treated with PEG, NaCl and drought was between 2.1 Umg-1 and 3.6 Umg-1, Of the transgenic lines had significantly higher enzyme activity than the corresponding non-transgenic lines. The relative conductivity of the membrane showed that the average relative conductivity of the transgenic plants treated with PEG, NaCl and drought was between 16.2% and 32.6%, and the relative conductivity of most of the transgenic lines under stress tolerance stress was significantly lower than that of the transgenic plants Corresponding control. The majority of transgenic BADH cabbage plants grew more vigorously under drought, salt stress and PEG stress than non-transgenic plants, indicating that the plant height increase of most transgenic lines was significantly higher than that of the control, indicating that the introduction of BADH gene can improve the transgenic cabbage plants Drought and salt tolerance. We obtained transgenic cabbage plants with significantly improved drought resistance and salt tolerance as germplasm materials for cultivating salt-tolerant and drought-resistant cabbage varieties.