Agrobacterium Tumefaciens-Mediated Transformation Of Strain Of Penicillium Janthinellum GXCR

It is beneficial to study the interaction between microorganisms and heavy mental in gene level. Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient tool for filamentous fungi because of high frequencies of transformation, stable DNA integration, random insertion and wide application that were difficult to transform with traditional methods.We previously isolated a Penicillium janthinellum strain GXCR with high resistance to many kinds of heavy mental salts and sensitive to benomyl. In this study, the genetic transformantion of GXCR mediated by Agrobacterium tumefaciens was successfully constructed. Make researching about its mechanism of resistance in gene level is possible. We constructed a binary vector named pG-Bn whose T-DNA harboring Benomyl resistance gene on the base of vector pGSA1252. Then, we transfer pG-Bn to LBA4404 by triparental cross and co-cultured with spores of GXCR. Now, we have obtained 367 transformants from that genetic system, PCR tests shows that 38 of 40 transformants can amplificate benomyl gene, the transformation efficiency is approximate 3.7 mutants /10 colonys. We choose 30 of 40 tranformations mentioned above at random, and we found that thirteen of them can't growth on the PDA which with CuSO₄ , and nine of them have obviously different in the concentration of CuSO₄ to wild-type, the rest are no visible different to wild-type. Southern blot analysis of transformants, the results show that 62.5 % of the mutants were T-DNA single copy intergration, that make the factional analysis of related genes via insertion mutation more easier. And also provided precious materials for the study of the genetic mechanism about GXCR resist to heavy mental.