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Construction and verification of a gene knockout system in the nematophagous fungus, Purpreocillium lilacinum (Hypocreales: Ophiocordycipitaceae)

文献类型： 外文期刊

作者： Yang, Fan 1 ; Abdelnabby, Hazem 1 ; Xiao, Yan-Nong 1 ;

作者机构： 1.Huazhong Agr Univ, Key Lab Plant Pathol Hubei Prov, Wuhan 430070, Hubei, Peoples R China

2.Henan Acad Agr Sci, Zhengzhou 450002, Henan Province, Peoples R China

3.Benha Univ, Fac Agr, Dept Plant Protect, Qalyubia 13736, Egypt

关键词： Purpreocillium lilacinum;Meloidogyne incognita;COX1 gene;ATMT;Gene knockout;Leucinostatins

期刊名称：APPLIED ENTOMOLOGY AND ZOOLOGY （ 2020影响因子：1.403； 五年影响因子：1.381 ）

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收录情况： SCI

摘要： Purpreocillium lilacinum (Thom) Luangsa-ard, Hywel-Jones, Houbraken and Samson (Hypocreales: Ophiocordycipitaceae) is a nematophagous fungus parasitizes eggs of plant parasitic nematodes, and a biocide in controlling nematode diseases of crops. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT), as an efficient gene knockout system, was applied as a first step to analyze the function of cytochrome oxidase subunit 1 (COX1) gene of the nematophagous fungus, P. lilacinum 36-1. A homologously recombined knockout vector was constructed using one-step PCR. The knockout vector contained two selectable markers coding for basta (bar) and carboxin (CBX) resistance genes. The new knockout system was performed with P. lilacinum 36-1 and the knocked out strain (Delta cox1) was successfully confirmed. The disruption of COX1 gene of P. lilacinum inhibited cytochrome c oxidase activity. Matrix-assisted laser desorption/ioniz-ation-time of flight mass spectrometry (MALDI-TOF MS) analysis of the fermentation liquid of P. lilacinum mutant strains revealed the absence of leucinostatins A and B, known as nematotoxin, from the Delta cox1 strain. Bioassay tests confirmed significant fading of Delta cox1 pathogenicity against Meloidogyne incognita (Kofoid and White) Chitwood, 1949 eggs and juveniles. Moreover, the growth rate and sporulation of Delta cox1 were significantly reduced compared to the wild type P. lilacinum 36-1. The phenotypic variation of knockout strain demonstrated that the knockout system is useful for investigating functional genes of P. lilacinum.

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[2]农杆菌介导的真菌T-DNA插入突变. 朱自贤,孙虎,薛保国. 2010

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