农杆菌介导的甜菜碱醛脱氢酶基因转化甘蓝的研究

Agrobacterium tumefaciens-mediated Transformation of Cabbage with Betaine Aldehyde Dehydrogenase Gene

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。

Cabbage （ Brassica oleracea vat. caphata ） is one of the most popular and widely cultivated vegetable crops in the world. In this paper, BADH （betaine aldehyde dehydrogenase） gene derived from spinach was transformed into the genome of cabbage line 03079 mediated by Agrobacterium t umefaciens. To establish the ideal transformation platform, the main factors which affect the transformation efficiency were optimized through the orthogonal design of L9（3^4 ）, including the type of infection medium, concentration of acetosyringone, the period of infection time and co-culture time. The results indicated that No. 9 was the best treatment combination, i. e, AA liquid medium was tile optimal infection medium for the cabbage transformation, and co-culturing for 2 days after infection （20 min） would be favorable for the transformation. If supplemented with 200μmol L^-1 acetosyringone in infection medium, the transformation efficiency would be improved and the transformed plant regenaration ratio reached at 54.26%. Based on this optimal transfonntion system, we obtained many transgenic plants, and PCR analysis using BADH gene primers and Southern blot analysis indicated that the BADH gene had been integrated into genome of cabbage. The BADH enzymes activity of transgenic plants were tested after treated with NaCl, drought-tolerance and PEG stress. The results showed that the average values of the activity of BADH enzymes, which varied from 2.1 U to 3.6 U per mg in transgenic plants, were 1 to 3 times higher than that of un-transgenie plants, and there was a significant difference between transgenie and un-transgenic plants using Duncan＇s Multiple Range Test. Furthermore, the average value of relative electronic conductivity （varying from 16.2 % to 32.6% ） of transgenic plants were significantly lower than that of un-transgenie plants, which indicated that the protection ability of membrane penetration was enhanced when BADH activity increasing and the stress resistance of the transgenic plants was improved by introduction BADH gene into cabbage. At the same time, most of the transgenie cabbage plants were vigrously growing under drought , salt and polyethylene glycol （PEG） stresses. The average height increasing percentage of transgenic plants were significantly higher than that of un-transgenic plants. Those results showed that the traits of drought or salt tolerance of transgenic cabbage plants were improved after transformed with BADH gene, and our transgenic plants would be used as fundamental germplasm for stress-tolerant breeding of cabbage.