《植物生理学报》 2013, 49(7): 689-699

共转化法获得HAK1基因高表达烟草提高植株钾吸收能力

谭颖^1,2,**, 秦利军^1,**, 赵丹^1,**, 赵杰宏^3,*, 赵德刚^1,2,*

贵州大学¹贵州省农业生物工程重点实验室, ²生命科学学院基因工程实验室, 贵阳550025; ³贵州省烟草科学研究院行业烟草分子遗传重点实验室, 贵阳550081. ** 同等贡献

通信作者：谭；E-mail: dgzhao@gzu.edu.cn, zhaojiehong@126.com；Tel: 0851-3863615

摘　要：

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。

关键词：烟草; 共转化; HAK1; 钾

收稿：2013-04-07   修定：2013-05-15

资助：贵州省科学技术基金“富钾基因发掘及无选择标记转基因烟草培育研究” [黔科合J字(2010)2251]、中国烟草总公司贵州省公司科技项目“抗TMV、富钾转基因烟草育种研究” [合同号(200910)]和国家转基因生物新品种培育重大专项“安全转基因技术研究” [2011ZX08010003-002]。

The Overexpression of HAK1 Gene Improved the Absorbing Ability for Potas-sium in Transgenic Tobacco by Co-Transformation Method

TAN Ying^1,2,**, QIN Li-Jun^1,**, ZHAO Dan^1,**, ZHAO Jie-Hong^3,*, ZHAO De-Gang^1,2,*

¹Guizhou Key Laboratory of Agro-Bioengineering, ²Laboratory of Genetic Engineering, College of Life Sciences, Guizhou Univer-sity, Guiyang 550025, China; ³The Key Laboratory of Molecular-Genetics on Industrial Tobacco, Research Institution of Tobacco Sciences in Guizhou Province, Guiyang 550081, China

Corresponding author: TAN Ying; E-mail: dgzhao@gzu.edu.cn, zhaojiehong@126.com; Tel: 0851-3863615

Abstract:

In order to improve the potassium-absorbing ability in tobacco plants, the leaf disks of tobacco variety K326 were transformed by using Agrobacterium tumefaciens mediated co-transformation method with two binary expression vectors, the pSH-TH and the pSH737. The pSH-TH contained tobacco high-affinity potassium transporter protein 1 gene (HAK1) and Nicotiana tabacum TMV resistance protein N (CN) gene and the pSH737 contained select maker genes. Thirty two T₀ generation transgenic tobacco plants were determined by GUS histochemical staining and PCR detection. Forty three T₁ marker-free transgenic tobacco plants with HAK1 and CN were selected by sequencing of PCR products and Southern blotting from T₁ generation population which was from T₀ plant selfing. Compared with wild type, the expression level of proton P-ATPase gene (NHA1) increased in transgenic plants, while the V-ATPase subunit G gene (VAG1) declined. Meanwhile, the expression level of the potassium outward-rectifying channel protein gene (TORK1) increased in transgenic lines except line T₁-87, and HAK1 increased in transgenic lines except line T₁-141. The expression level of HAK1 in line T₁-51, T₁-87 and T₁-100 was about 3 folds of wild type. Besides, root activity, root ATPase activity, root cation exchange capacity and K⁺ content in trangenic plants were higher than those in wildtypes with significant difference. The results indicated that over expression of HAK1 improved the ability of enriching potassium in tobacco plants, and these safely transgenic tobacco plants without select-marker gene can be used as the parent materials for breeding high quality tobacco variety.

Key words: tobacco; co-transformation; HAK1; potassium