Relationship between expression level of hygromycin B-resistant gene and Agrobacterium tumefaciens-mediated transformation efficiency in Beauveria bassiana JEF-007.

AIMS

Agrobacterium tumefaciens-mediated transformation (AtMT) 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 AtMT efficiency was investigated to improve the transformant selection.

METHODS AND RESULTS

Ten B. bassiana isolates were grown on 800 μg ml 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, pCeg (gpdA promoter based) and pCambia-egfp (CaMV 35S promoter based), were constructed to evaluate the promoters on the expression of HygB resistance gene (hph) at 100, 150 and 200 μg ml HygB medium. Eight days after the transformation, wild type, AtMT/pCeg and AtMT/pCambia-egfp colonies were observed on 100 μg ml HygB, but significantly larger numbers of colonies were counted on AtMT/pCeg plates. At higher HygB concentration (150 μg ml ), only AtMT/pCeg colonies were further observed, but very few colonies were observed on the wild type and AtMT/pCambia-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 AtMT transformation, and the hph expression level in AtMT/pCeg colonies was higher than that of AtMT/pCambia-egfp colonies.

CONCLUSIONS

In the HygB-susceptible B. bassianaJEF-007, gpdA promoter works better than CaMV 35S promoter in the expression of HygB resistance gene at 150 μg ml HygB, consequently improving the selection efficiency of putative transformants.

SIGNIFICANCE AND IMPACT OF THE STUDY

These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters.