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通过农杆菌介导,将仅含钾转运体蛋白基因HAK1和烟草TMV抗性蛋白基因CN的pSH-TH表达载体和仅含选择标记基因GUS::NPTII的pSH737表达载体共转化烟草K326,从抗性转化苗中筛选出32株T0代转基因植株,自交分离后经筛选获得43株无选择标记的T1代转基因烟株,Southern blotting分析和PCR产物测序结果证明HAK1及CN功能基因已整合到烟草基因组中,且不含标记基因。转基因植株中的钾转运相关基因表达分析表明,细胞质膜P-H+-ATPase基因NHA1和烟草无机焦磷酸酶基因NVP1表达上调,烟草液泡膜V-H+-ATPase基因VAG1表达下调;而烟草钾外流通道蛋白基因TORK1表达在株系T1-87中下调,其余株系均上调;HAK1表达量除了在株系T1-141中下调外,在其他所有株系中均上调,其中株系T1-12、T1-51、T1-87和T1-100的HAK1表达量比野生型高3倍以上。株系T1-51、T1-87和T1-100的根系活力、ATPase活性、阳离子交换量(CEC)及钾含量均显著高于野生型,表明钾离子转运体基因HAK1超量表达显著提高了烟草植株的富钾能力,所获得的无标记的安全转基因株系可作为优质烟叶品质培育的亲本材料。
Through Agrobacterium-mediated transformation, pSH-TH expression vector containing only the potassium transporter protein HAK1 and tobacco TMV resistance protein CN and the pSH737 expression vector containing only the selectable marker GUS :: NPTII were co-transformed into tobacco K326, A total of 32 T0 transgenic plants were selected from the transformed plants and 43 isolates with T1 generation were screened by selfing. Southern blotting and PCR products showed that the HAK1 and CN functional genes were integrated into tobacco In the genome, there is no marker gene. The analysis of potassium transport related genes in transgenic plants showed that NHA1 and NVP1 were up-regulated and the expression of VAG1 in tobacco tonoplast was down-regulated. The potassium outflow channel The expression of TORK1 protein was down-regulated in T1-87 and all other strains were up-regulated. The expression of HAK1 was up-regulated in all the other strains except T1-141, -51, T1-87 and T1-100 HAK1 expression three times higher than the wild-type. The root activity, ATPase activity, cation exchange capacity (CEC) and potassium content in T1-51, T1-87 and T1-100 lines were significantly higher than those in wild type, indicating that overexpression of potassium ion transporter gene HAK1 significantly increased tobacco The potassium-rich ability of plants, the obtained unlabeled safe transgenic lines can be used as parent material to cultivate high-quality tobacco.