转G10aroA棉花株系的获得及分子生物学鉴定

Molecular Biology Identification of Transgenic Cotton Lines Expressing Exogenous G10aroA Gene

【目的】培育具有抗草甘膦除草剂的棉花材料具有极其重要的意义。利用农杆菌介导法在棉花中转入编码EPSPS酶的抗草甘膦除草剂基因G10aroA,通过体细胞愈伤诱导组织培养技术获得能够稳定遗传的转基因棉花株系材料。【方法】首先,利用不同草甘膦抗性筛选条件比较分析不同棉花受体材料的愈伤诱导效率;其次,以R15材料作为受体,利用含有G10aroA的农杆菌侵染下胚轴切段,在进行体细胞愈伤诱导的组织培养过程中通过草甘膦抗性筛选获得棉花再生植株,对获得的棉花再生植株进行纯合繁育。在此基础上,利用PCR扩增检测证实外源G10aroA在转基因植株中能够稳定遗传;利用RT-PCR分析其外源基因在转基因植株不同组织中的转录水平进行研究、并进一步利用Western-blot对转基因植株中外源蛋白的表达进行分析。【结果】在优化的草甘膦筛选条件下,以草甘膦浓度为2.5 mmol·L-1的抗性条件进行棉花愈伤诱导筛选并获得棉花再生植株;利用特异引物进行PCR检测结果表明,在检测的全部再生植株中,扩增得到1.8 kb预期大小目标条带的阳性株系32株,其中,收获的27个株系的外源目标基因能够在T0、T1转基因植株中稳定遗传;对G10aroA在转基因株系L12、L14的不同组织中的转录表达进行定量RT-PCR分析表明,外源G10aroA在转基因棉花植株的不同组织中表达具有差异,相对表达量高低依次为茎、苞叶、叶和花;另外,蛋白检测结果进一步表明,该外源基因能够在转基因株系L7、L12和L14中植株中正常表达为预期46 kD的EPSPS蛋白。【结论】通过农杆菌介导法转化外源抗草甘膦基因G10aroA,在草甘膦抗性条件下进行棉花体细胞诱导的组织培养,成功获得转外源G10aroA的棉花再生株系,并通过分子生物学方法研究证实外源G10aroA能够在T0、T1转基因株系中稳定遗传、转录以及表达。

[Objective] It is of great value to develop glyphosate-resistant cotton in China. To obtain the glyphosate-tolerant transgenic cotton line, a new GlOaroA gene encoding a 5-enolpyruvylshikimate- 3-phosphate synthase （EPSPS） was transformed into the cotton by Agrobacterium-mediated transformation method. The glyphosate-tolerant transgenic cotton lines can be obtained by somatic callus tissue culture techniques. [ Method] Firstly, the callus induction efficiency of recipient cotton was tested underdifferent concentrations of glyphosate. Secondly, GlOaroA was transformed into the hypocotyl segments of cotton line R15 using Agrobacterium-mediated method. The transgenic cotton plants were regenerated from callus tissue culture under selection of glyphosate. The T cotton plant seeds were obtained by self-pollination of To plants. The GlOaroA gene in the transgenic cotton genome was confirmed by PCR, and the transcripts of the GlOaroA gene in the transgenic cotton lines were analyzed by RT-PCR. The expression of EPSPS encoded by GlOaroA gene was detected by Western blot. [ Result] Glyphosate concentration of 2.5 mmol.L-1 was selected by comparative analysis for tissue culture. Under this condition, the GlOaroA gene was introduced into R15 cotton and thirty-two positive cotton lines were regenerated. These To transgenic lines were strictly self-pollinated. The GlOaroA gene was confirmed by PCR detection of GlOaroA in twenty-seven transgenic lines of T generation. The results suggested that the exogenous gene was stably inherited in all of twenty-seven T1 transgenic plants. Quantitative real-time PCR analysis revealed that the exogenous GlOaroA gene was expressed varied among different tissues in transgenic line L12 and L14. The order of relative expression level from high to low was stems, bracts, leaves and flowers. In addition, the EPSPS protein of 46 kD size encoded by G10aroA gene was detected in the leaves of transgenic line L7, L 12 and L 14. [ Conclusion ] The G10aroA gene has been integrated into genomic DNA of the cotton lines via Agrobacterium-mediated method. The transcripts and protein of GlOaroA were both detected in the T progeny. Thus the transgenic lines may be used for cotton breeding research of glyphosate-tolerant cotton.