转G10aroA棉花株系的获得及分子生物学鉴定

doi:10.3864/j.issn.0578-1752.2014.06.002

Abstract

Abstract: 【Objective】It is of great value to develop glyphosate-resistant cotton in China. To obtain the glyphosate-tolerant transgenic cotton line, a new G10aroA 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 under different concentrations of glyphosate. Secondly, G10aroA 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 T1 cotton plant seeds were obtained by self-pollination of T0 plants. The G10aroA gene in the transgenic cotton genome was confirmed by PCR, and the transcripts of the G10aroA gene in the transgenic cotton lines were analyzed by RT-PCR. The expression of EPSPS encoded by G10aroA 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 G10aroA gene was introduced into R15 cotton and thirty-two positive cotton lines were regenerated. These T0 transgenic lines were strictly self-pollinated. The G10aroA gene was confirmed by PCR detection of G10aroA in twenty-seven transgenic lines of T1 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 G10aroA 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，L12 and L14. 【Conclusion】 The G10aroA gene has been integrated into genomic DNA of the cotton lines via Agrobacterium-mediated method. The transcripts and protein of G10aroA were both detected in the T1 progeny. Thus the transgenic lines may be used for cotton breeding research of glyphosate-tolerant cotton.

Key words: cotton (Gossypium hirsutum L.) , glyphosate , G10aroA , EPSPS

WANG Xia-1, 2 , 4 , MA Yan-Bin-2, WU Xia-2, SHEN Zhi-Cheng-3, LIN Chao-Yang-3, LI Peng-Bo-2, SUN Xuan-2, WANG Xin-Sheng-2, LI Yan-E-2, LI Gui-Quan-1. Molecular Biology Identification of Transgenic Cotton Lines Expressing Exogenous G10aroA Gene[J].Scientia Agricultura Sinica, 2014, 47(6): 1051-1057.

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