Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (1): 80-89.doi: 10.3864/j.issn.0578-1752.2014.01.009

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning and Resistance Verification of a Target Gene of Glyphosate from Klebsiella pneumoniae S001

 ZHANG  Chun-1, WU  Dan-Dan-1, 2 , FENG  Li-1, TIAN  Xing-Shan-1, GUO  Ai-Ling-2   

  1. 1.Institute of Plant Protection, Guangdong Academy of Agricultural Sciences/Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Guangzhou 510640;
    2.College of Food Science & Technology, Huazhong Agricultural University, Wuhan 430000
  • Received:2013-04-28 Online:2014-01-01 Published:2013-09-30

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Abstract: 【Objective】Due to the widespread use of glyphosate in agricultural production, glyphosate has become the preferred object on transgenic crops resistant to herbicides. Finding glyphosate resistance genes is a primary issue in the study of transgenic crops resistant to glyphosate. There are various kinds of microorganisms in nature with rich genetic resources, so this study intends to screen and identify high glyphosate-resistance bacterium strains from field soil samples of Guangdong area, then clone the target gene of glyphosate from the strain and test its glyphosate-resistance level, in order to obtain high glyphosate resistance gene resources for research of glyphosate resistant transgenic crops.【Method】The gradient dilution glyphosate selected culture method in the isolation of high glyphosate-resistance bacterium from field soil samples of Guangdong area was applied. Identification of the strain species according to the microscopic observation, gram staining and 16 S rDNA sequence analysis results. Using RT-PCR method to clone the strain’s target gene of glyphosate, and analyse the basic characteristics of aroAS001 sequences by sequence alignment and phylogenetic tree. The aroAS001 variant performed site directed mutagenesis by overlapping PCR method to obtain aroAS001-mut gene fragments. The aroAS001 and aroAS001-mut fragments were transferred into defective Escherichia coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. 【Result】 A high glyphosate-resistance bacterium strain was isolated, and it was identified as Klebsiella pneumoniae by morphological and molecular biology methods, named kpS001 strain. The target gene of glyphosate from kpS001 strain named aroAS001 was cloned. Sequence analysis showed that the amino acid sequence of the gene encoded had a typical Class I EPSPS features, and there was a single amino acid different from with another K. pneumoniae strain’s aroA. After obtained the aroAS001-mut fragments by overlap PCR, aroAS001 and aroAS001-mut fragments were transferred into the defective E. coli DH5α/△aroA strains, respectively, to detect the resistant levels of glyphosate. Compared with the control strains, the recombinant strains containing aroAS001 and aroAS001-mut were able to grow normally in the culture medium containing less than 200 mmol?L-1 glyphosate, however, as glyphosate concentration increasing, the growth state of the recombinat strains gradually suppressed, while the concentration of glyphosate increased to 350 mmol?L-1, the growth was completely suppressed. 【Conclusion】The K. pneumoniae S001 is a high glyphosate-resistance bacterium, the target gene of glyphosate aroAS001 belongs to the Class I EPSPS and shows a significant glyphosate-resistance characteristics, it could be used as potential gene materials in transgenic glyphosate-resistant crop studies.

Key words: Klebsiella pneumoniae , 5-enolpyruvoylshikimate-3-phosphate synthase , aroAS001 , glyphosate-resistance

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