Nai Y S, Lee M R, Kim S, Lee S J, Kim J C, Yang Y T, Kim J S
Department of Agricultural Biology, College of Agriculture & Life Sciences, Chonbuk National University, Jeonju, Korea.
Department of Biotechnology and Animal Science, College of Bioresources, National Ilan University, I-Lan, Taiwan, ROC.
J Appl Microbiol. 2017 Sep;123(3):724-731. doi: 10.1111/jam.13529. Epub 2017 Jul 31.
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.
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.
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.
These results provide useful information for determining AtMT effectiveness in B. bassiana isolates, particularly antibiotic susceptibility and the role of promoters.
根癌农杆菌介导的转化（AtMT）是一种用于产生昆虫病原性球孢白僵菌转化体的有效方法。然而，一些菌株能在含有潮霉素B（HygB）的选择培养基上生长，这降低了假定转化体的筛选效率。在本研究中，对潮霉素B抗性基因启动子与AtMT效率之间的关系进行了研究，以提高转化体的筛选效率。
将10株球孢白僵菌分离株在800μg/ml HygB培养基上培养，但只有JEF-006、-007和-013对该抗生素敏感。特别是，JEF-007表现出最明显的剂量依赖性敏感性。构建了两种不同的Ti质粒，即基于gpdA启动子的pCeg和基于CaMV 35S启动子的pCambia-egfp，以评估在100、150和200μg/ml HygB培养基上启动子对潮霉素B抗性基因（hph）表达的影响。转化8天后，在100μg/ml HygB培养基上观察到野生型、AtMT/pCeg和AtMT/pCambia-egfp菌落，但在AtMT/pCeg平板上计数的菌落数量明显更多。在较高的HygB浓度（150μg/ml）下，仅进一步观察到AtMT/pCeg菌落，而在野生型和AtMT/pCambia-egfp平板上观察到的菌落很少。对假定的转化体进行PCR、RT-PCR和qRT-PCR，以研究T-DNA插入率和基因表达水平。结果，>80%的菌落显示AtMT转化成功，且AtMT/pCeg菌落中hph的表达水平高于AtMT/pCambia-egfp菌落。
在对HygB敏感的球孢白僵菌JEF-007中，在150μg/ml HygB条件下，gpdA启动子在潮霉素B抗性基因表达方面比CaMV 35S启动子表现更好，从而提高了假定转化体的筛选效率。
这些结果为确定AtMT在球孢白僵菌分离株中的有效性提供了有用信息，特别是抗生素敏感性和启动子的作用。
