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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志  2020, Vol. 40 Issue (9): 11-17    DOI: 10.13523/j.cb.2004031
研究报告     
农杆菌介导海洋草酸青霉转化体系及聚酮合酶Pks生物学功能*
孟晓琳,庞锡明,王洁()
华南农业大学食品学院 广州 510642
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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摘要:

为研究南海柳珊瑚共附生草酸青霉SCSGAF0023的聚酮合酶(PKS)生物学功能,采用农杆菌介导法构建草酸青霉SCSGAF0023的Pks敲除株ΔPks,比较野生菌株及ΔPks的生长发育及环境适应性差异。以草酸青霉SCSGAF0023分生孢子为受体,p0380-hygB为双元载体,成功实现草酸青霉SCSGAF0023的遗传转化。结果表明:农杆菌浓度为OD600=0.5,在200μmol/L 乙酰丁香酮(AS)诱导下与107个/ml草酸青霉SCSGAF0023孢子于25℃共孵育时转化效率最高。基于上述转化体系,成功获得Pks敲除株ΔPks,并首次证实Pks正向调控草酸青霉SCSGAF0023产孢,但不影响其对环境的适应性。这为进一步系统研究真菌PKSs及聚酮化合物对真菌生长发育与环境适应性的影响提供素材。

关键词: 草酸青霉农杆菌介导转化聚酮合酶生长发育环境适应性    
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
收稿日期: 2020-04-22 出版日期: 2020-10-12
ZTFLH:  Q819  
基金资助: * 国家自然科学基金青年科学基金(31600060);广东省科技创新战略专项资金-重点领域研发计划(2018B020206001);广东省科技创新战略专项资金-重点领域研发计划(2020B020226008)
通讯作者: 王洁     E-mail: wangjielangjing@126.com
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引用本文:

孟晓琳,庞锡明,王洁. 农杆菌介导海洋草酸青霉转化体系及聚酮合酶Pks生物学功能*[J]. 中国生物工程杂志, 2020, 40(9): 11-17.

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.

链接本文:

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

Primers Paired sequences (5'-3')* Purpose
hygB-F/R AAAAAGAGCTCGACGTTAACTGATATTGAAGGAGC /
AAAAACTCGAGAACCCAGGGGCTGGTGAC
Cloning the full length of hygB (1 898bp)
L1/L2 AAAAACCCGGGTGGCATTGATTCCAGAGACT /
AAAAAGGATCCCCAGGACAAGTCACGAAGGT
Cloning Pks 5'-end (1 979bp)
R1/R2 AAAAAAGATCTTACAGTGGTCTCCTTTCCCG /
AAAAAACTAGTACATTGTGCCACTCATCCAG
Cloning Pks 3'-end (2 217bp)
D1/D2 AGCCTATCTCAGAGCAACGC / CCAGAGAGCACCAAGAGCAG PCR detecting Pks
表1  用于草酸青霉Pks基因操作的引物.
图1  p0380-hygB阳性转化株PCR鉴定图
图2  孵育条件对草酸青霉SCSGAF0023遗传转化的影响
图3  Pks的保守区域及其基因敲除株鉴定
图4  Pks敲除后草酸青霉在不同环境条件下的生长
图5  Pks敲除影响草酸青霉SCSGAF0023产孢
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