Agrobacterium-mediated Transformation and the Functions of Pks in Marine-derived Penicillium oxalicum

doi:10.13523/j.cb.2004031

China Biotechnology

2020, Vol. 40

Issue (9): 11-17 DOI: 10.13523/j.cb.2004031

Agrobacterium-mediated Transformation and the Functions of Pks in Marine-derived Penicillium oxalicum

MENG Xiao-lin,PANG Xi-ming,WANG Jie(

)

College of Food Science, South China Agricultural University, Guangzhou 510642, China

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Abstract

To investigate the functions of polyketide synthases (PKS) in Penicillium oxalicum SCSGAF0023, a marine gorgonian coral-associated fungus, the method of Agrobacterium-mediated transformation in P. oxalicum SCSGAF0023 was constructed and used to obtain the disruption mutants of Pks by homologous recombination. The roles of Pks in P. oxalicum SCSGAF0023 were characterized by multi-phenotypic analyses under normal and stressful conditions. An efficient genetic transformation system of P. oxalicum SCSGAF0023 was established under the condition of p0380-hygB being binary vector and conidia being the recipient. The transformation efficiency was the highest when 10⁷ conidia/ml was co-cultivated with Agrobacterium tumefaciens AGL-1 with the OD₆₀₀ of 0.5 under the induction of 200μmol/L acetoyringone (AS) at 25℃. Based on the Agrobacterium-mediated transformation, the disruption mutants of Pks were obtained, and it was the first reported that Pks positively regulated the conidiation but did not affect the environmental adaptation in P. oxalicum SCSGAF0023, which will be helpful for further investigation on the relationships between PKSs and fungal growth, development and environmental adaptation.

Key words： Penicillium oxalicum Agrobacterium-mediated transformation Polyketide synthase Fungal growth and development Environmental adaptation

Received: 22 April 2020 Published: 12 October 2020

ZTFLH:

Q819

Corresponding Authors: Jie WANG E-mail: wangjielangjing@126.com

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	Xiao-lin MENG
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Cite this article:

MENG Xiao-lin,PANG Xi-ming,WANG Jie. Agrobacterium-mediated Transformation and the Functions of Pks in Marine-derived Penicillium oxalicum. China Biotechnology, 2020, 40(9): 11-17.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2004031 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I9/11

Table 1 Paired primers used for the manipulation of P. oxalicum Pks

Fig.1 PCR identification for the positive transformants of p0380-hygB M: 250bp DNA ladder

Fig.2 The influences of incubation conditions on the transformation efficiency in P. oxalicum (a)-(d)The effects of AS concentrations, co-cultivation concentrations of AGL-1 and conidia, and co-cultivation temperature on the transformation efficiency,respectively

Fig.3 Features of Pks and identification of its disruption mutants (a) Main domains of Pks typical for PKS family KS: Ketosynthase; AT: Acyltransferase; DH: Dehydratase; MT: Methyltransferase; KR: Ketoreductase; PP: Phosphopantetheine attachment site for acyl carrier protein or peptidyl carrier protein (b) Diagram for the disruption of Pks L1/L2 and R1/R2: Paired primers used for cloning the 5' and 3' regions of Pks; D1/D2: Paired primers used for PCR detection of disrupted target gene (c) Identifying the disruption mutants by PCR

Fig.4 Changes in fungal growth caused by the disruption of Pks (a) Clononies grown on the PDA with different pH values (b) The relative growth inhibition caused by the addition of Congo red (CR), NaCl, KCl, H₂O₂ and MnCl₂

Fig.5 Pks disruption affected the conidiation of P. oxalicum SCSGAF0023 The bar marked with an asterisk within each group differs significantly from those unmarked (Tukey’s HSD, P<0.05). Error bars: SD from three repeated assays


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