采用农杆菌介导的遗传转化方法,将来自枯草杆菌的果聚糖蔗糖转移酶基因(SacB)导入银腺杨,以提高杨树对水分胁迫的抗性.以来自无菌培养的叶片为外植体,通过大约1 000个叶盘与农杆菌LBA4404共培养,将植物双元表达载体pKP中SacB基因导入银腺杨基因组,经卡那霉素筛选后,共获得102株卡那霉素抗性植株.经PCR特异性扩增和Southern点杂交分析,证明其中97株再生植株基因组DNA中整合了SacB基因.对其中的62个无性系进行RT-PCR分析,结果表明SacB基因在其中的50个无性系中获得表达.温室生长观察表明,转基因无性系外部形态与对照相比没有稳定的显著差异,少数部分转基因无性系的生长明显受到抑制,其他转基因无性系生长正常.这些转基因无性系的获得为培育抗旱转基因杨树奠定了基础.%The present study describes the transfer of a Bacillus subtilis SacB gene, with vacuolar targeting signal sequences and driven by constitutive promoters, by Agrobacterium tumefaciens into poplar( Populus alba × P. glandulosa). From about 1 000 leaf discs used for transformation, 102 Km-resistant plants were obtained, and 97 proved to be true transgenic plants. The transgenic nature of these plants was confirmed by PCR amplification and Southern dot hybridization. The expression of the chimeric SacB genes in transgenic plants was confirmed by RT-PCR. The performance of some transgenic lines maintained under a normal watering regime was evaluated over 5 months in greenhouse. These plants showed no significant stable morphological differences from the untransformed plants. The growth of some plants was apparently inhibited, while most of the plants grew at least as well as the control when water is non-limiting. This material may be the basis for obtaining a more drought-resistant poplar.
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