关键词: 陆地棉, 抗草甘膦棉花, 拷贝数, 融合位点, 分子特征

Abstract:

【Objective】 To obtain the transgenic cotton by Agrobacterium-mediated method was the purpose using the new gene G10aroA with high glyphosate tolerance in our laboratory. Meanwhile, it was necessary to provide molecular characteristics of genomic integration of the exogenous gene for breeding utilization in future. 【Method】 The Agrobacterium-mediated method was used for transgenic cotton plants obtained via tissue culture with glyphosate herbicide. Western blot was utilized to detect the expression of exogenous proteins in different organs of transgenic cotton R1-3. The number of loci in cotton genomes were evaluated by Southern blot for detecting integration of the exogenous sequence from the pCAMBIA1300 construct. The flanking sequence near the insertion site was amplified by TAIL-PCR, which the extractions of DNA were cloned and sequenced. The location of the chromosome for the flanking sequences were compared and analyzed on the website of NCBI blast. 【Result】 Regenerated R1-3 cotton plants were successfully obtained by tissue culture depending on glyphosate screening. the specific protein coded by exogenous G10aroA gene could be detected normally via Western blotting in the leaves, bracts, flowers and stems separately, and the size of the exogenous protein around 46 kDa were also observed in this experiment. In addition, the result of Southern-blot confirmed that the exogenous fragment containing G10aroA sequences was single integration in the genome of transgenic cotton R1-3, in which the bands digested severally by KpnⅠ and EcoRⅠ endonucleases were distinctly observed near the position of 6 557 bp and 4 316 bp strips on the nylon membrane respectively. The analysis of flanking sequence alignment for the integration site was predicted to be located on the 11th chromosome of either cotton A or D genome, and the left and right boundaries of the insertion site were further located between 47 525 303 and 47 525 449 of the chromosomes. In addition, the specific identification for the fusion site showed that the target band of approximately 300 bp for testing left border junction, and a specific target band can be amplified about 600 bp for testing the right border fusion site. 【Conclusion】 In this study, we obtained R1-3 transgenic cotton plants that also have exhibited stable genetic characteristics of glyphosate resistance during the process of self-crossing breeding. The protein coded by G10aroA gene was about the size of 46 kDa that could be detected in different tissues of transgenic cotton R1-3 plant. Furthermore, the exogenous fragment including G10aroA gene was identified with a single location by southern blot in the cotton genomes, and the integration site was located at the 11th chromosome. The results of comparative analysis were predicted that a nucleotide sequence about 146 bp length was deleted at the integration of the genome.

Key words: Gossypium hirsutum L., glyphosate-tolerance cotton, copy number, integration site, molecular characterization