禾谷镰孢核孔蛋白基因FgNup42的功能分析

doi:10.3864/j.issn.0578-1752.2021.09.007

摘要/Abstract

摘要：

【目的】核孔蛋白Nup42在真核生物基因表达调控以及mRNA加工运输等生物学过程中发挥着重要作用,本研究旨在分析禾谷镰孢（Fusarium graminearum）中核孔蛋白基因FgNup42在病原菌生长发育、逆境胁迫、致病和产毒等生物学过程中的功能。【方法】通过融合PCR（double-joint PCR）和酵母空隙修复（gap repair）技术分别构建FgNup42基因敲除和回补载体,再利用PEG介导的原生质体转化的方法获得基因敲除突变体ΔFgNup42和回补体ΔFgNup42-C。观察测定基因敲除突变体ΔFgNup42在营养生长、无性繁殖和有性生殖过程中的变化,同时测定突变体ΔFgNup42对渗透、杀菌剂以及细胞壁胁迫因子的敏感性。将突变体ΔFgNup42进行田间麦穗和室内玉米花丝接种试验明确其致病力情况。通过液相色谱-串联质谱（LC-MS/MS）检测ΔFgNup42的产毒能力,同时利用qRT-PCR比较分析参与单端孢霉烯族毒素生物合成的7个TRI在野生型PH-1和基因敲除突变体ΔFgNup42中的相对表达量。【结果】表型测定发现,基因敲除突变体ΔFgNup42的生长速率只有野生型PH-1的50%,菌落边缘菌丝分枝变多且致密。显微观察分生孢子形成情况,发现敲除突变体ΔFgNup42相较野生型PH-1分生孢子产量降低了85.45%,并且隔膜数在0—2的分生孢子比例明显增多。有性生殖诱导结果显示ΔFgNup42的有性生殖能力增强,较野生型产生了更多的子囊壳。突变体ΔFgNup42对渗透胁迫因子NaCl和KCl,细胞壁胁迫因子刚果红,以及杀菌剂戊唑醇和氰烯菌酯的敏感性减弱。致病力分析发现敲除基因FgNup42后菌体在麦穗和玉米花丝上的致病力严重降低。此外,与野生型相比,ΔFgNup42中毒素DON、3ADON和15ADON的合成量明显减少。【结论】核孔蛋白基因FgNup42在禾谷镰孢生长发育、抵御逆境以及致病和产毒过程中发挥着重要作用。

关键词: 禾谷镰孢, FgNup42, 生长发育, 环境胁迫, 致病力

Abstract:

【Objective】The nucleoporin Nup42 plays important roles in gene expression regulation, nuclear mRNA processing and export. The objective of this study is to analyze the function of the nucleoporin gene FgNup42 in growth and development, stress, pathogenicity and mycotoxins production of Fusarium graminearum. 【Method】Targeted gene deletion and mutant complementation constructs were generated using the double-joint PCR and yeast gap repair methods, respectively. Then the gene deletion mutant ΔFgNup42 and complemented strain ΔFgNup42-C were obtained by PEG-mediated protoplast transformation. The vegetative growth, asexual and sexual reproduction of the gene disrupted mutant ΔFgNup42 were examined. Moreover, the sensitivity of ΔFgNup42 to osmotic stress, fungicides and cell wall damaging agent were investigated. Pathogenicity of the mutant ΔFgNup42 was assayed by point-inoculated the flowering wheat heads and incubation on maize silks. Mycotoxins produced by ΔFgNup42 were quantified using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The relative transcription levels of 7 TRIs involved in trichothecene mycotoxins biosynthesis were determined by qRT-PCR between PH-1 and ΔFgNup42. 【Result】Phenotype analysis showed that the growth rate of ΔFgNup42 was 50% of that of wild type PH-1, but more dense in hyphal branching. Microscopic examination revealed that the conidiation of ΔFgNup42 mutant decreased by 85.45% compared with the wild type PH-1, and percentage of conidia with septum range 0 to 2 was significantly increased. The ΔFgNup42 mutant produced more perithecia than the wild type strain in mating cultures suggesting improved sexual reproduction. The mutant ΔFgNup42 displayed increased tolerance to osmotic stress NaCl and KCl, fungicides tebuconazole and phenamacril, also cell wall damaging agent Congo red. Pathogenicity assays exhibited that disruption of gene FgNup42 caused a dramatic reduction in pathogenicity by inoculated the flowering wheat heads and maize silks. Furthermore, the amount of mycotoxins DON, 3ADON and 15ADON produced by ΔFgNup42 were significantly lower than that produced by wild type. 【Conclusion】The nucleoporin gene FgNup42 plays a key role in fungal growth and development, pathogenesis, mycotoxins production and responses to various environmental stresses in F. graminearum.

Key words: Fusarium graminearum, FgNup42, growth and development, environmental stress, pathogenicity

张承启,廖露露,齐永霞,丁克坚,陈莉. 禾谷镰孢核孔蛋白基因FgNup42的功能分析[J]. 中国农业科学, 2021, 54(9): 1894-1903.

ZHANG ChengQi,LIAO LuLu,QI YongXia,DING KeJian,CHEN Li. Functional Analysis of the Nucleoporin Gene FgNup42 in Fusarium graminearium[J]. Scientia Agricultura Sinica, 2021, 54(9): 1894-1903.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.09.007

https://www.chinaagrisci.com/CN/Y2021/V54/I9/1894

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引物 Primer	序列 Sequence (5′-3′)
Nup42-up-F	GCGCTCTCAAGAGAGTCACCG
Nup42-up-R	CAAAATAGGCATTGATGTGTTGACCTCCCGCGGTAAGCTCTGGCTACCT
Nup42-down-F	CTCGTCCGAGGGCAAAGGAATAGAGTAGCATAGGTAGGTAGGTATCTAC
Nup42-down-R	TTGATTCCAGCCTCCAGCTG
HPH-F	GGAGGTCAACACATCAATGCCTATT
HPH-R	CTACTCTATTCCTTTGCCCT
Nup42-nest-F	CGAGGGCCATGTCATTCCTG
Nup42-nest-R	GAGTGAGATTCTAGGATCGT
Nup42-ID-F	GTATCTACTATGGCGATATTGC
Nup42-ID-R	GTATCCAACCAATCAGCGTGTC
Nup42-GFP-F	ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTGCGCTCTCAAGAGAGTCACCG
Nup42-GFP-R	CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACAAAGTCCCACAATGTGCATTC
ID-Nup42-GFP-F	CCGCAAGCTAACAATCCATTC
ID-GFP-R	GTCAGCTTGCCGTAGGTGGCA
Tri1-RT-F	GATGTTCCTTCTCGACAGCGT
Tri1-RT-R	CACTGGTCGAAGATAGCTGG
Tri3-RT-F	TGTTACGATCAATGGCTTGG
Tri3-RT-R	TCCTCGTTGTAGTTTGCATCA
Tri4-RT-F	ACGTGTGGCTACTCAGGAGAA
Tri4-RT-R	TGGAATTGCCTTGGGGTA
Tri6-RT-F	AAATGCCCATTCCCTAGTTG
Tri6-RT-R	ATCTCGCATGTTATCCACCCT
Tri7-RT-F	TACCGTCGTCTTCAAAACCA
Tri7-RT-R	ACGCCAATGGTGTTCACAAA
Tri8-RT-F	ATATAACGGTACCCCCAGATG
Tri8-RT-R	TGTTTGTAGGACACTTCCGGT
Tri14-RT-F	AACTCCCGTTGTGATCAAGCA
Tri14-RT-R	AACAGTAATGTTGGCACCGT
actin-RT-F	CCACGTCACCACTTTCAACT
actin-RT-R	TGCTTGGAGATCCACATTTG