转几丁质酶基因烟草遗传稳定性研究

Study on the inheritance stability of chitinase expressing transgenic tobacco

从转几丁质酶基因烟草株系(NPTII基因是选择标记基因)对卡那霉素的抗性,及外源几丁质酶基因表达蛋白的活性(WesternBlot检测)两个方面对转基因株系的遗传稳定性进行了分析研究。在卡那霉素抗性检测试验中,检测了13个T2 转基因株系,其中9个株系被检测种子10 0 %对卡那霉素(10 0mg/L)具有抗性;T3 和T4转基因株系各检测了10个,10 0 %被检测种子在含卡那霉素10 0mg/L的MS培养基上长成了烟苗。WesternBlot检测了11个T2 株系,检测结果表明,6个株系被检测植株10 0 %含有外源几丁质酶蛋白,2个株系被检测植株80 %含有外源几丁质酶蛋白,2个株系被检测植株5 0 %含有外源几丁质酶蛋白,1个株系被检测植株19 .0 5 %含有外源几丁质酶蛋白。在温室内将转基因烟草与未转基因烟草密植在一起,种植2代,烟株开花期模仿自然风力对烟株吹风,进行了转基因烟草是否可以通过花粉进行基因漂移的研究。研究结果表明,在自然风媒条件下未发现转基因烟草可进行基因漂移。

Whether the exo-chitinase gene can be stably inherited in the transgenic tobacco was studied. Firstly, using MS medium containing Kanamycin (100mg/L) geminated the T_2 ,T_3 and T_4 generation transgenic tobacco seeds to test whether the transgenic tobacco seeds contain the NPT II gene, which is a selectable marker gene. Secondly analyzed the protein of transgenic tobacco leaves by Western Blot to test whether the exo-chitinase gene still express protein in the T_2 and T_3 generations. In the Km resistance detection experiment, 11 T_2 transgenic lines were tested. Among them 9 lines 100% seeds have resistance to Km. 10 T_3 and 10 T_4 transgenic lines were tested; all tested seeds have resistance to Km. In the western blot experiment, 11 T_2 transgenic tobacco lines were tested. And the experimental results showed that all tested plants in 6 lines contain the chitinase protein; 80% tested plants of 2 lines contains exo-chitinase protein; 50% tested plants of 2 lines contains exo-chitinase protein; and in 1 line 19.05% tested plants contain exo-chitinase protein. Meanwhile whether the transgene can cause gene flow through wind pollination was also studied. In the green house under the mimic natural wind condition, both transgenic and untransgenic tobacco plants are planted together for two generations. The seeds of each plant were collected separately, and geminated in the MS medium-containing Km (100mg/L). No untransgenic tobacco seeds have resistance to Km were found.