Xie Long-Xu, Li Yun-Feng, Xu Pei-Lin
State Key Laboratory for Biocontrol, Zhongshan University, Guangzhou 510275, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2004 Apr;30(2):173-8.
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 micromol/L. Globular embryos were induced and then developed by culturing on MSB (MS salts+B(5) 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 T(0) transgenic plants. 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. Western blot analysis showed that a 48-kD band of was detected in all T(0) transgenic plants. There was no apparent corelation between copy numbers and the expression level of the aroAM12 gene. Greenhouse screening for glyphosate resistance was performed to test 65 independent T(0) 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 T(1) generation was measured by spraying with Roundup, the numbers of glyphosate resistance and sensitive phenotypes showed Mendelian segregation ratio.
在之前一项研究中，利用交错延伸过程，使用来自鼠伤寒沙门氏菌和大肠杆菌的aroA基因产生了一种对草甘膦具有抗性的突变体aroAM12。在本文中，我们构建了携带aroAM12基因的载体pGRA1300，其包含拟南芥EPSPS的转运肽，在CaMV35S启动子的控制下，并用作棉花（陆地棉）转化的选择标记。通过用根癌农杆菌共转化下胚轴片段，在含有草甘膦的培养基上直接筛选，获得了对草甘膦抗性增强的转基因棉花。在含有0.1 mg/L 2,4-D、0.1 mg/L KT、500 mg/L头孢噻肟和60 μmol/L草甘膦的MS基本培养基上进行抗草甘膦愈伤组织的再生。通过在补充有1 g/L天冬酰胺和2 g/L谷氨酰胺但不含激素的MSB（MS盐+B5维生素）培养基上培养40天来诱导球形胚并使其发育。然后将发育的幼苗取出并在MS培养基上培养。约20天后，将生根良好的芽移栽到土壤中。PCR分析表明，aroAM12基因存在于所有T0转基因植物中。Southern杂交进一步证实了aroAM12基因整合到棉花基因组DNA中，结果表明转基因植物携带一或两个aroAM12基因拷贝。Western杂交分析表明，在所有T0转基因植物中均检测到一条48-kD的条带。aroAM12基因的拷贝数与表达水平之间没有明显的相关性。通过以相当于9.317 kg/ha农达的剂量（每5天一次，共三次）喷洒水悬浮液，对65株独立的T0植物进行温室草甘膦抗性筛选。15天后，与未转化的对照植物相比，对这些植物进行表型检查。在这些条件下，观察到用pGRA1300转化的植物对草甘膦表现出高抗性，而对照植物全部死亡。通过喷洒农达来测定T1代的草甘膦抗性，草甘膦抗性和敏感表型的数量呈现孟德尔分离比。
