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我们将从地衣芽孢杆菌(Bacillus licheni-formis)克隆的约1.68kb的耐高温α-淀粉酶基因构建成表达载体,并转入根癌农杆菌。以马铃薯栽培品种“杂交荷兰7号”块茎圆盘为外植体,按本实验室建立起的再生实验系统及杨美珠等的方法进行转化。采取共培养、芽的诱导、芽的选择再生三步方法获得抗性芽。将抗性芽通过先诱导生根壮苗,再进行卡那霉素筛选,最后再诱导生根的方法得到可能的转基因植株。对部分可能的转基因植株按改进的王广立等的PCR简单快速鉴定转基因植物的方法进行检测,株号102001、102607、110402均可见到特异性片段的存在。参照张振清及王福荣等的方法对这些植株进行耐高温α-淀粉酶活力测定,这些植株具有相对较强的耐高温α-淀粉酶活性。实验结果表明,耐高温α-淀粉酶基因可能已转入上述植物基因组中,并获表达。
We constructed an approximate 1.68 kb thermostable alpha-amylase gene cloned from Bacillus licheni-formis as an expression vector and transferred it into Agrobacterium tumefaciens. The potato cultivar “Hybrid Netherlands 7” tuber disc was used as explant, and the regeneration experiment system established by this laboratory and Yang Meizhu et al. Were used for transformation. Take co-culture, bud induction, bud regeneration of three-step selection method to obtain resistant buds. The resistant shoots were induced first by rooting and then by kanamycin screening. Finally, the induced transgenic plants were obtained by inducing rooting. Some of the possible transgenic plants were detected by the method of simple and rapid identification of transgenic plants by modified Wang Guangli et al. The presence of specific fragments can be seen in strain Nos. 102001, 102607 and 110402. The heat-resistant α-amylase activity of these plants was determined by the methods of Zhang Zhenqing and Wang Furong. These plants have relatively high temperature-resistant α-amylase activity. The experimental results show that the high-temperature α-amylase gene may have been transferred into the plant genome and expressed.