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Acta Agron Sin ›› 2014, Vol. 40 ›› Issue (07): 1190-1196.doi: 10.3724/SP.J.1006.2014.01190

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning of New Herbicide Resistant Gene in Soil Metagenomics and the Generation of Transgenic Rice Plants

WANG Yun-Peng1,2,MA Jing-Yong1,MA Rui1,MA Jian 1,*,LIU Wen-Guo1,2,*   

  1. 1Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, China; 2Jilin Academy of Agricultural Sciences, Changchun 130033, China
  • Received:2014-01-26 Revised:2014-04-16 Online:2014-07-12 Published:2014-05-16
  • Contact: 马建, E-mail: majian19790106@163.com; 刘文国, E-mail: liuwenguo168@163. com

Abstract:

The EPSPS, EC 2.5.1.19 (5-enolpyruvylshikimate-3-phosphate synthase) is considered as one of the crucial enzyme in the shikimate pathway for the biosynthesis of essential aromatic amino acids and secondary metabolites in plant. It is also proved as a specific target of broad spectrum herbicide glyphosate. In this research, a new glyphosate resistant EPSPS gene named soilEPSPS was isolated by screening the metagenomic library. The sequence analysis suggested that soilEPSPS consists of 1404 bp and encodes a polypeptide of 467 amino acids that are not involved in the amino acid sequence protected by the published patentsions. The result of prokaryotic expression confirmed that soilEPSPS had a better ability of glyphosate resistance than EPSPS CP4 gene. Fusing the gene and the Rubisco SSU signal peptide constructed the plant expressing vector with the actin promoter driven, and transformation of rice mediated by Agrobacterium tumefaciens. PCR and Southern analyses showed that a total of 26 transgenic plants were obtained and three of them were single copy insertion events. Resistant experiment showed that the homozygous T2 plants in these events could tolerate the spray of 500 mmol L-1 glyphosate solution. This study laid a foundation for breeding transgenic herbicide resistant rice varieties.

Key words: Metagenomics, Rice, Agrobacterium tumefaciens, Genetic transformation

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