To establish and optimize the genetic transformation technology of Penicillium marneffei by Agrobacterium tumefaciens-mediated transformation

Abstract

Abstract:

Objective To construct and optimize the condition of Agrobacteriumtumefaciens-mediated transformattechology in Penicillium marneffei.Method A.tumefaciens transformed by Calcium chloride with pDHt/SK::pyrG plasmids insert pyrG gene into SPM4 (pyrG^-,niaD^-),medium without uracil screening positive transformat,Using PCR verification restructuring.It aimed at finding out the optimal protocol by changing some important effecting factors including A.tumefaciens strains,concentration,transformat medium,co-cultivation temperature,period,AS.The positive transformant growed well in the media without uracil,and PCR analysis showed that T-DNA was inserted into the transformants.Result The transformation efficiency was about 300 transformants/10⁶ spores by optimizing.The optimal condition for AGL-1,concentration of A.tumefaciens OD₆₀₀=0.8,the AS concentration of 200 μmol/L,without nitrocellulose filters,co-culture 48 h in 25℃.Conclusion A.tumefaciens-mediated transformattechology in P.marneffei.was successfully established,simplified and optimized.The method can be used for PM gene function research.

Key words: Penicillium marneffei, Agrobacterium tumefaciens, transformation

CLC Number:

R379.9

LI Xin-lei, LI Xin, CAO Cun-wei. To establish and optimize the genetic transformation technology of Penicillium marneffei by Agrobacterium tumefaciens-mediated transformation[J]. Chinese Journal of Mycology, 2015, 10(2): 88-91.

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