根癌农杆菌介导的转aroAM12基因棉花植株的草甘膦抗性

Glyphosate-resistant Cotton (Gossypium hirsutum L.) Transformed with aroAM12 Gene via Agrobacterium tumefaciens

以中棉35无菌苗下胚轴为外植体,采用农杆菌介导法将含有通过基因优化技术获得的草甘膦抗性突变基因aroAM12导入棉花中。以aroAM12为选择标记,利用草甘膦直接筛选获得65棵再生植株。PCR和Southernblot分析表明,经过草甘膦筛选出的T0代植株均整合有aroAM12基因。Western blot分析表明整合进的aroAM12基因得到了有效表达,且不同植株之间的表达不尽相同。大棚喷洒的实验结果表明T0代转化植株具有很高的草甘膦抗性。对T1代棉花的草甘膦抗性遗传分析表明,aroAM12基因以盂德尔方式遗传。

A mutant, aroAM12, exhibiting resistance to glyphosate produced in a previous study using the staggered extension process with aroA genes from Salmonella typhimurium and Eschrichia coli. In this paper, we constructed a vector pGRA1300 carrying aroAM12 gene, comprising transit peptide of Arabidopsis EPSPS, under the control of the CaMV35S promoter and used as selectable marker for cotton plant (Gossypium hirsutum L.) transformation. Transgenic cottons with increased resistance to glyphosate were obtained by cotransformtion of hypocotyl segments with Agrobacterium tumefaciens and selected directly on medium containing glyphosate. Regeneration of glyphosate-resistant calli was carried out on a MS basic medium containing 2,4-D 0.1 mg/L, KT 0.1 mg/L, cefotaxime 500 mg/L and glyphosate 60 μmol/L. Globular embryos were induced and then developed by culturing on MSB (MS salts+B5 vitamins) medium supplemented with asparagine 1 g/L and glutamine 2 g/L, but not containing hormone, for 40 d. The developed plantlets were then removed and cultured on an MS medium. After about 20 d, the deeply-rooted shoots were in soil. PCR analysis showed that the aroAM12 gene was present in all T0 transgenic plants (Fig.3). The integration of the aroAM12 gene in the genomic DNA of cotton was further confirmed by Southern blot, which showed that the transgenic plants carried one or two copies of the aroAM12 genes (Fig.4). Western blot analysis showed that a 48-kD band of was detected in all T0 transgenic plants. There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene (Fig.5), Greenhouse screening for glyphosate resistance was performed to test 65 independent T0 plants by spraying (three times) with an aqueous suspension at a dose corresponding to 9.317 kg/ha of Roundup (once every 5 d). After 15 d, phenotype examination was carried out of the plants in comparison with untransformed control plants. Under these conditions, it was observed that the plants transformed with pGRA1300 showed high resistance to glyphosate whereas the control plants were all killed. The glyphosate resistance of T1 generation was measured by spraying with Roundup, the numbers of glyphosate resistance and sensitive phenotypes showed Mendelian segregation ratio.