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Relationship between expression level of hygromycin B‐resistant gene and Agrobacterium tumefaciens ‐mediated transformation efficiency in Beauveria bassiana JEF ‐007
Author(s) -
Nai Y.S.,
Lee M.R.,
Kim S.,
Lee S.J.,
Kim J.C.,
Yang Y.T.,
Kim J.S.
Publication year - 2017
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13529
Subject(s) - agrobacterium , biology , beauveria bassiana , transformation (genetics) , hygromycin b , agrobacterium tumefaciens , microbiology and biotechnology , gene , genetics , botany , biological pest control
Aims Agrobacterium tumefaciens ‐mediated transformation (At MT ) is an effective method for generation of entomopathogenic Beauveria bassiana transformants. However, some strains grow on the selective medium containing hygromycin B (HygB), which reduces the selection efficiency of the putative transformants. In this work, a relationship between HygB resistance gene promoter and At MT efficiency was investigated to improve the transformant selection. Methods and Results Ten B. bassiana isolates were grown on 800 μ g ml −1 HygB medium, but only JEF ‐006, ‐007 and ‐013 showed susceptibility to the antibiotics. Particularly, JEF ‐007 showed the most dose‐dependent susceptibility. Two different Ti‐Plasmids, pC eg ( gpdA promoter based) and pC ambia‐egfp (Ca MV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene ( hph ) at 100, 150 and 200 μ g ml −1 HygB medium. Eight days after the transformation, wild type, At MT / pC eg and At MT / pC ambia‐egfp colonies were observed on 100 μ g ml −1 HygB, but significantly larger numbers of colonies were counted on At MT / pC eg plates. At higher HygB concentration (150 μ g ml −1 ), only At MT / pC eg colonies were further observed, but very few colonies were observed on the wild type and At MT / pC ambia‐egfp plates. Putative transformants were subjected to PCR , RT ‐ PCR and qRT ‐ PCR to investigate the T‐ DNA insertion rate and gene expression level. Consequently, >80% of colonies showed successful At MT transformation, and the hph expression level in At MT / pC eg colonies was higher than that of At MT / pC ambia‐egfp colonies. Conclusions In the HygB‐susceptible B. bassiana JEF ‐007, gpdA promoter works better than Ca MV 35S promoter in the expression of HygB resistance gene at 150 μ g ml −1 HygB, consequently improving the selection efficiency of putative transformants. Significance and Impact of the Study These results provide useful information for determining At MT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters.