花生疮痂病菌蓝光受体EaWC 1基因克隆及生物信息学分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0937

摘要/Abstract

摘要：

光是真菌感知和适应环境的重要信息载体,调控多种生理生化过程。痂囊腔菌素（Elsinochromes,ESC）是花生疮痂病菌重要的毒力因子,蓝光对其具有正调控作用,为了进一步揭示光调控ESC机制,开展了花生疮痂病菌蓝光受体基因EaWC 1克隆、生物信息学分析和表达模式研究。结果表明,蓝光受体基因EaWC 1全长为3 261bp,具有一个完整的开放阅读框,编码长度为1 086个氨基酸的蛋白质,相对分子质量11.95 kD,理论等电点为8.81,具有核定位信号,为稳定亲水性蛋白。qPCR定量分析表明,EaWC 1基因表达受到蓝光诱导,其表达模式与ESC毒素含量呈显著正相关。研究结果对于阐明花生疮痂病菌蓝光受体生物功能,揭示ESC毒素生物合成光调控机制和调控网络奠定了理论基础。

关键词: Elsinoë arachidis, 蓝光受体, 基因克隆, 生物信息学分析, 表达分析

Abstract:

Light is an important information carrier for fungi to perceive and adapt to the environment factors,regulate a variety of physiological and biochemical processes. Elsinochromes（ESC）is an important virulence factor of Elsinoë arachidis causing peanut scab. In order to further reveal mechanism of light regulation on ESC production,gene cloning,bioinformatics analysis and expression of the blue-light receptor gene EaWC 1 were conducted. The results showed that the blue light receptor gene EaWC-1 had a complete reading frame（ORF）with 3 261 bp in length that encoded a protein of 1 086 amino acids. The molecular weight of the protein was 11.95 kD,the theoretical isoelectric point was 8.81. Subcellular localization analysis showed that EaWC-1 was localized in nucleus and was a hydrophilic protein. qPCR analysis indicated that the expression of EaWC 1 gene was positively regulated by blue light,and its expression pattern was significantly and positively correlated with ESC toxin accumulation. This result would provide a theoretical foundation for elucidating the biological functions of blue light receptors of E. arachidis and revealing the photo-regulation mechanism and regulatory network of ESC toxin biosynthesis.

Key words: Elsinoë arachidis, blue-light receptor, gene cloning, bioinformatics analysis, expression analysis

李洋, 张晓天, 朴静子, 周如军, 李自博, 关海雯. 花生疮痂病菌蓝光受体EaWC 1基因克隆及生物信息学分析[J]. 生物技术通报, 2022, 38(5): 93-99.

LI Yang, ZHANG Xiao-tian, PIAO Jing-zi, ZHOU Ru-jun, LI Zi-bo, GUAN Hai-wen. Cloning and Bioinformatics Analysis of Blue-light Receptor EaWC 1 Gene in Elsinoë arachidis[J]. Biotechnology Bulletin, 2022, 38(5): 93-99.

图/表 10

表1 WC 1同源基因序列信息

Table 1 Sequence information of WC 1 homologous gene

种名Species	GenBank ID
Elsinoe fawcettii	KAF4549824.1
Elsinoe australis	TKX21094.1
Elsinoe ampelina	KAF2224433.1
Alternaria alternata	QBZ93284.1
Cercospora zeae maydis	AEH41590.1
Pseudocercospora musae	KXT04460.1
Dothistroma septosporum	EME40353.1
Trichoderma atroviride	AAU14171.1
Neurospora crassa	CAA63964.2
Aspergillus nidulans	AAP47230.1
Mucor circinelloides	CAJ13843.2
Phycomyces blakesleeanus	ABB77846.1
Hortaea werneckii	RMY69450.1

图1 EaWC 1基因PCR扩增

Fig. 1 PCR amplification of EaWC 1 gene

图2 WC-1 蛋白质结构域分析

Fig.2 Protein domain analysis of WC-1

图3 WC-1蛋白氨基酸序列比对

Fig. 3 Amino acid sequence alignment of WC-1 protein

图4 EaWC-1蛋白序列系统进化树

Fig. 4 Phylogenetic tree of EaWC-1 protein sequence

图5 蛋白疏水性

Fig. 5 Protein hydrophobicity

图6 编码蛋白磷酸位点分析

Fig.6 Analysis of phosphate sites of encoded protein

图7 蛋白二三级结构预测

Fig. 7 Secondary and tertiary structure prediction of protein

图8 病菌培养特性

Fig. 8 Culture characteristics of E. arachidis

图9 EaWC 1基因表达分析及ESC含量

Fig.9 EaWC 1 expression analysis and ESC production light quality

参考文献 30

[1]	Yu Z, Fischer R. Light sensing and responses in fungi[J]. Nat Rev Microbiol, 2019, 17(1):25-36. doi: 10.1038/s41579-018-0109-x URL
[2]	王艺, 韦小丽. 不同光照对植物生长、生理生化和形态结构影响的研究进展[J]. 山地农业生物学报, 2010, 29(4):353-359, 370.
	Wang Y, Wei XL. Advance on the effects of different light environments on growth, physiological biochemistry and morphostructure of plant[J]. J Mt Agric Biol, 2010, 29(4):353-359, 370.
[3]	Fischer R, Aguirre J, Herrera-Estrella A, et al. The complexity of fungal vision[J]. Microbiol Spectr, 2016, 4(6):FUNK-0020-2016. DOI: 10.1128/microbiolspec.funk-0020-2016. doi: 10.1128/microbiolspec.funk-0020-2016
[4]	Corrochano LM. Fungal photoreceptors:sensory molecules for fungal development and behaviour[J]. Photochem Photobiol Sci, 2007, 6(7):725-736. doi: 10.1039/b702155k URL
[5]	Kertesz-Chaloupková K, Walser PJ, Granado JD, et al. Blue light overrides repression of asexual sporulation by mating type genes in the Basidiomycete Coprinus cinereus[J]. Fungal Genet Biol, 1998, 23(1):95-109. pmid: 9514695
[6]	Sanz C, Rodríguez-Romero J, Idnurm A, et al. Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism[J]. PNAS, 2009, 106(17):7095-7100. doi: 10.1073/pnas.0900879106 URL
[7]	Zhu P, Zhang C, Xiao H, et al. Exploitable regulatory effects of light on growth and development of Botrytis cinerea[J]. J Plant Pathol, 2013, 95(3):509-517.
[8]	Ma YJ, Sun CX, Wang JW. Enhanced production of hypocrellin A in submerged cultures of Shiraia bambusicola by red light[J]. Photochem Photobiol, 2019, 95(3):812-822. doi: 10.1111/php.13038 URL
[9]	郭明敏, 杨涛, 卜宁, 等. 大型真菌光受体及其功能研究进展[J]. 菌物学报, 2015, 34(5):880-889.
	Guo MM, Yang T, Bu N, et al. Photoreceptor and its functions in macrofungi:a review[J]. Mycosystema, 2015, 34(5):880-889.
[10]	Fuller KK, Loros JJ, Dunlap JC. Fungal photobiology:visible light as a signal for stress, space and time[J]. Curr Genet, 2015, 61(3):275-288. doi: 10.1007/s00294-014-0451-0 pmid: 25323429
[11]	Losi A, Gärtner W. The evolution of flavin-binding photoreceptors:an ancient chromophore serving trendy blue-light sensors[J]. Annu Rev Plant Biol, 2012, 63:49-72. doi: 10.1146/annurev-arplant-042811-105538 pmid: 22136567
[12]	Crosthwaite SK, Dunlap JC, Loros JJ. Neurospora wc-1 and wc-2:transcription, photoresponses, and the origins of circadian rhythmicity[J]. Science, 1997, 276(5313):763-769. pmid: 9115195
[13]	Cheng P, Yang YH, Gardner KH, et al. PAS domain-mediated WC-1/WC-2 interaction is essential for maintaining the steady-state level of WC-1 and the function of both proteins in circadian clock and light responses of Neurospora[J]. Mol Cell Biol, 2002, 22(2):517-524. doi: 10.1128/MCB.22.2.517-524.2002 pmid: 11756547
[14]	Röhrig J, Kastner C, Fischer R. Light inhibits spore germination through phytochrome in Aspergillus nidulans[J]. Curr Genet, 2013, 59(1/2):55-62. doi: 10.1007/s00294-013-0387-9 URL
[15]	Fuller KK, Ringelberg CS, Loros JJ, et al. The fungal pathogen Aspergillus fumigatus regulates growth, metabolism, and stress resistance in response to light[J]. mBio, 2013, 4(2):e00142-13. DOI: 10.1128/mbio.00142-13. doi: 10.1128/mbio.00142-13
[16]	Kim H, Son H, Lee YW. Effects of light on secondary metabolism and fungal development of Fusarium graminearum[J]. J Appl Microbiol, 2014, 116(2):380-389. doi: 10.1111/jam.12381 pmid: 24176027
[17]	Silva F, Torres-Martínez S, Garre V. Distinct white collar-1 genes control specific light responses in Mucor circinelloides[J]. Mol Microbiol, 2006, 61(4):1023-1037. doi: 10.1111/j.1365-2958.2006.05291.x URL
[18]	Cervantes-Badillo MG, Muñoz-Centeno T, Uresti-Rivera EE, et al. The Trichoderma atroviride photolyase-encoding gene is transcriptionally regulated by non-canonical light response elements[J]. Febs J, 2013, 280(15):3697-3708. doi: 10.1111/febs.12362 pmid: 23721733
[19]	Pruβ S, Fetzner R, Seither K, et al. Role of the Alternaria alternata blue-light receptor LreA(white-collar 1)in spore formation and secondary metabolism[J]. Appl Environ Microbiol, 2014, 80(8):2582-2591. doi: 10.1128/AEM.00327-14 URL
[20]	Kim H, Ridenour JB, Dunkle LD, et al. Regulation of stomatal tropism and infection by light in Cercospora Zeae-maydis:evidence for coordinated host/pathogen responses to photoperiod?[J]. PLoS Pathog, 2011, 7(7):e1002113. doi: 10.1371/journal.ppat.1002113 URL
[21]	张观红. 灰霉菌蓝光受体基因bcwc1的功能分析[D]. 上海: 华东师范大学, 2014.
	Zhang GH. Functional analysis of white collar-1 Bcwc1 in Botrytis cinerea[D]. Shanghai: East China Normal University, 2014.
[22]	Daub ME, Chung KR. Photoactivated Perylenequinone Toxins in Plant Pathogenesis.[M]// B. edsPlantRelationships. TheMycota(A Comprehensive Treatise on Fungi as Experimental Systems for Basic and Applied Research). Berlin: Springer, 2009:201-219
[23]	Liao HL. Molecular and genetic determination of the role of elsinochrome toxins produced by Elsinoe fawcettii causing citrus scab[D]. Florida: University of Florida, 2008.
[24]	焦文莉. 花生疮痂病菌光生物学及全基因组序列分析[D]. 沈阳: 沈阳农业大学, 2019.
	Jiao WL. Photobiology and whole genome analysis of Elsinoë arachidis[D]. Shenyang: Shenyang Agricultural University, 2019.
[25]	Ballario P, Vittorioso P, Magrelli A, et al. White collar-1, a central regulator of blue light responses in Neurospora, is a zinc finger protein[J]. EMBO J, 1996, 15(7):1650-1657. pmid: 8612589
[26]	Froehlich AC, Liu Y, Loros JJ, et al. White collar-1, a circadian blue light photoreceptor, binding to the frequency promoter[J]. Science, 2002, 297(5582):815-819. pmid: 12098706
[27]	Lokhandwala J, Hopkins HC, Rodriguez-Iglesias A, et al. Structural biochemistry of a fungal LOV domain photoreceptor reveals an evolutionarily conserved pathway integrating light and oxidative stress[J]. Structure, 2015, 23(1):116-125. doi: S0969-2126(14)00368-2 pmid: 25533487
[28]	Zoltowski BD, Vaccaro B, Crane BR. Mechanism-based tuning of a LOV domain photoreceptor[J]. Nat Chem Biol, 2009, 5(11):827-834. doi: 10.1038/nchembio.210 pmid: 19718042
[29]	Amaral PP, Dinger ME, Mercer TR, et al. The eukaryotic genome as an RNA machine[J]. Science, 2008, 319(5871):1787-1789. doi: 10.1126/science.1155472 pmid: 18369136
[30]	Estrada AF, Avalos J. The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation[J]. Fungal Genet Biol, 2008, 45(5):705-718. doi: 10.1016/j.fgb.2007.12.003 URL