MGV1 Dependent Transcripts(MDT1)Gene in Fusarium Graminearum is Involved in Conidiation and Vegetative Growth

doi:10.13523/j.cb.2002002

China Biotechnology

2020, Vol. 40

Issue (8): 10-18 DOI: 10.13523/j.cb.2002002

MGV1 Dependent Transcripts(MDT1)Gene in Fusarium Graminearum is Involved in Conidiation and Vegetative Growth

PENG Hai-li,HOU Zhan-ming(

)

College of Life Science and Technology, Inner Mongolia Normal University, Hohhot 010022, China

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Abstract

Background: Fusarium graminearum is the major pathogen of Fusarium head blight of wheat and other grain crops. To understand mechnism of pathogenesis of the pathogen, molecular analysis has widely carried out during the past dacades. The differential gene expression (DGE) analysis for MGV1 deletion mutant revealed that the MDT1 gene was significantly down-regulated in MGV1 knock-out mutant, suggesting that the gene was involved in the MGV1 pathway. Go annotation given out that the MDT1 gene was involved in the ATP binding and protein dimerization activity. Objective: In order to identify the function of gene, MDT1(MGV1 dependent transcripts)Gene was isolated and characterized. Methods: The split-marker strategy was applied to construct the deletion cassette of the gene and the phenotype and pathogenicity of the deletion mutant were assayed by conventional method. Results: MDT1 gene deletion mutant significantly decreased the amount of conidia and produced less perithecia than that of the wild type, indicating that the gene was essential for the asexual and sexual reproduction. Vegetative growth of the mutant greatly reduced in solid medium. The mutant also was hypersensitive to cell wall degrading enzyme and formed swollen top of the mycelium and fractured hyphea at 32℃in liquid medium, revealing that the gene was related to the cell wall integration in Fusarium graminearum. The complement test confirmed the phenotypic changes of the MDT1 gene deletion. Conclusion: The results demonstrated that MDT1 gene in Fusarium graminearum was involved in conidiation and vegetative growth.

Key words： Fusarium graminearum MDT1 Split-marker Gene deletion Condiation

Received: 06 February 2020 Published: 10 September 2020

ZTFLH:

Q819

Corresponding Authors: Zhan-ming HOU E-mail: houzhm@imnu.edu.cn

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Cite this article:

PENG Hai-li,HOU Zhan-ming. MGV1 Dependent Transcripts(MDT1)Gene in Fusarium Graminearum is Involved in Conidiation and Vegetative Growth. China Biotechnology, 2020, 40(8): 10-18.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2002002 OR https://manu60.magtech.com.cn/biotech/Y2020/V40/I8/10

Table 1 Primers used in this study

Fig.1 The diagram of principle of the pZPH1

Fig.2 Construction and screening for △FGSG_07836 (a) PCR amplification of upstream and downstream sequences of MDT1 gene(1: PCR product of upstream of MDT1; 2: PCR product of downstream of MDT1) (b) PCR amplification of upstream and downstream sequences of hph gene(1: pCB1003 plasmid DNA as template, HYG/F and HY/R as upstream primers out of hph upstream sequences; 2: pCB1003 plasmid DNA as template, YG/F and HYG/R as downstream primers out of hph downstream sequences) (c) Overlapping PCR products for MDT1 Split-marker recombination(1: FGSG_07836 (1F/2R) PCR products with PCB1003 (HYG/F and HY/R) PCR products as templates; MDT1 gene upstream of the gene 3' end primer FGSG_07836 1F and hph upstream of the 5' end primer HY/R; 2: FGSG_07836 (3F/4R) PCR products with PCB1003 (YG/F and HYG/R) PCR products as templates; MDT1 gene downstream of the gene 5' end of the primer FGSG_07836 4R and hph downstream of the 3' end of the primer YG/F) (d) PCR positive screening though HYG/F and HYG/R as positive primer (e) PCR positive screening though FGSG_07836 1F and HY/R as positive primer (f) PCR negative screening though FGSG_07836 N1F and FGSG_07836 N2R as positive primer M: 1kb plus DNA Ladder;d, e, f: 1~5: Genomic DNA of △FGSG_07836 as template;6: Genomic DNA of the wild type as template

Fig.3 Construction and screening for pZPH1 plasmid (a) pZWH1 plasmid DNA digested by BamH I (b) Purification of product digested by BamH I (c) 17 white single colony PCR screens (d) Colony 2 plasmid DNA (e),(f) Colony 2 plasmid DNA PCR ladder (e: FGSG_07836 N1F / FGSG_07836 N2R as primer, f: FGSG_07836 com1F/FGSG_07836 com2R as primer) (g) Colony 2 plasmid DNA digested by EcoRI M: 1kb plus DNA Ladder

Fig.4 Screening of FGSG_07836 C PCR (a) FGSG_07836 N1F/ FGSG_07836 N2R as primer (b) NeoF/NeoR as primer (c) HYG/F-HYG/R as primer M: 1kb plus DNA Ladder (real-times); 1~3: Genomic DNA of transformant 1~3 as template

Fig.5 Colonies and conidial morphology of different strains (a) Colony morphology of PH-1, △FGSG_07836 and FGSG_07836C cultured on 25℃,4d TCC (b) The mycelium growth rate of PH-1, △FGSG_07836 and FGSG_07836C on 25℃,5d TCC (c) Conidia morphology of PH-1, △FGSG_07836 and FGSG_07836C in 25℃, 175r/min CMC, Magnification 16×402.3.2 产孢量变化分别取10μl菌株PH-1,△FGSG_07836和FGSG_07836C孢子(CMC培养基中),通过血球计数板计数,其分生孢子量分别为:菌株PH-1 12×10⁵个/ml;△FGSG_07836 6×10⁵个/ml,FGSG_07836C 11×10⁵个/ml。通过统计分析,菌株PH-1,FGSG_07836C与△FGSG_07836的分生孢子量有显著差异,菌株PH-1与FGSG_07836C无显著差异(P<0.01,P<0.05,表2)。

Table 2 Statistical analyses on conidia, colony diameter and infect diameter of different strains

Fig.6 Comparison of protoplasts, hyphae and sexual hybrids of different strains (a) The release of protoplasing of wild type PH-1, △FGSG_07836 and FGSG_07836C after incubation for 1h (b) The condition of hyphal growth of PH-1, △FGSG_07836 and FGSG_07836C in 25℃ TB3 (c) The condition of hyphal growth of PH-1, △FGSG_07836 and FGSG_07836C in 32℃ TB3 Magnification 16×40 (d) Sexual reproduction of strain PH-1, △FGSG_07836 and FGSG_07836C in 19d carrots cultured

Fig.7 Infection experiment of different strains (a) Strain PH-1, △FGSG_07836 and FGSG_07836C pores infect tomato (b) Infection assays of PH-1, △FGSG_07836 and FGSG_07836C on maize


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