扩展青霉erg4的生物信息学、亚细胞定位及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0141

摘要/Abstract

摘要：

麦角甾醇是真菌细胞质膜的特有组分,在真菌生长发育中具有重要作用。erg4是参与麦角甾醇生物合成最后一步反应的基因,但扩展青霉中该基因的功能未知。本文通过RT-PCR方法克隆了扩展青霉3个erg4（erg4A、erg4B 和 erg4C）基因的 CDS全长,对基因结构、编码蛋白的跨膜螺旋和亲疏水性进行了生物信息学分析,通过融合绿色荧光蛋白定位的方法进行了亚细胞定位,测定了3个基因在不同生长发育阶段、不同培养基状态以及黑暗和蓝光条件下的表达差异。扩展青霉erg4A、eerg4B 和 erg4C的 CDS全长分别为1 476 bp、1 491 bp和 1 596 bp,分别编码491、496和 531个氨基酸;编码蛋白均属于跨膜蛋白,且表现出疏水性。绿色荧光蛋白与内质网红色荧光探针染色共定位结果显示,Erg4A、Erg4B和Erg4C均定位于内质网。erg4A、erg4B 和erg4C在孢子阶段、孢子萌发阶段及成熟菌丝阶段的表达水平存在显著差异,其中,erg4A在3个阶段的表达量无明显变化,而erg4B和 erg4C的表达量均显著上调,以erg4B的上调幅度最为明显。erg4A在 CY液体和固体培养条件下的表达量无显著变化,erg4B和erg4C在CY液体培养条件下的表达量显著高于固体培养,以erg4B的上调幅度最为明显。erg4A和 erg4B在蓝光条件下的表达量显著高于黑暗条件,以erg4B的上调幅度最为明显。erg4C对蓝光条件不敏感。扩展青霉Erg4A、Erg4B和 Erg4C均定位于内质网,erg4A、erg4B和 erg4C在不同生长发育阶段、固体和液体以及黑暗与蓝光培养条件下的表达存在较大差异,其中,以erg4B的响应最为活跃。

关键词: 扩展青霉erg4, 生物信息学分析, 亚细胞定位, 表达差异

Abstract:

Ergosterol,a fungus-specific component in cell plasma membranes,plays an essential role in fungal growth and development. The erg4 gene encodes the catalytic enzyme for the last step of ergosterol synthesis,whereas,the function of this gene in Penicillium expansum is unknown. In this study,the full-length CDSs of erg4A,erg4B and erg4C in P. expansum were cloned by RT-PCR,and their genes’ structures,transmembrane domain and hydrophobicity of the erg4 encoded proteins were analyzed via bioinformatics. The subcellular localizations of 3 proteins were also analyzed by fusion green fiuorescent protein method. In addition,the expression differences of 3 genes in different growth stages,different medium conditions,and dark and blue light conditions were measured. The full-length CDSs of erg4A,erg4B and erg4C were 1 476 bp,1 491 bp and 1 596 bp,respectively,and the number of encoded amino acids were 491,496 and 531,respectively. The encoded proteins were all transmembrane proteins and presented hydrophobicity. The co-localization results of green fluorescent protein and endoplasmic reticulum fluorescent probe showed that Erg4A,Erg4B and Erg4C were all located in endoplasmic reticulum. There were significant differences in the expressions of erg4A,erg4B and erg4C in spore,spore germination and mature mycelium stages. The expression of erg4A had no significant variation in the three stages,while the expressions of erg4B and erg4C were significantly up-regulated,and that of erg4B was the most obvious. There was no significant variation in the expression of erg4A under CY liquid and solid culture conditions. The expressions of erg4B and erg4C under CY liquid culture conditions were significantly higher than those under solid culture conditions,and the up-regulation of erg4B was the most obvious. The expressions of erg4A and erg4B under blue light were significantly higher than those under dark conditions,and the up-regulation of erg4B was the most obvious. erg4C was not sensitive to blue light condition. In conclusion,Erg4A,Erg4B and Erg4C were all located in the endoplasmic reticulum,and the expressions of erg4A,erg4B and erg4C vaied significantly in different growth stages,solid and liquid cultural conditions,and dark and blue light culture conditions,among which erg4B was the most active.

Key words: Penicillium expansum erg4, bioinformatics analysis, subcellular localization, expression differences

韩占红, 宗元元, 张学梅, 王斌, PRUSKY Dov, 毕阳. 扩展青霉erg4的生物信息学、亚细胞定位及表达分析[J]. 生物技术通报, 2021, 37(12): 60-70.

HAN Zhan-hong, ZONG Yuan-yuan, ZHANG Xue-mei, WANG Bin, PRUSKY Dov, BI Yang. Bioinformatics,Subcellular Localization and Expression Analysis of erg4 in Penicillium expansum[J]. Biotechnology Bulletin, 2021, 37(12): 60-70.

图/表 16

表1 erg4A、erg4B和erg4C基因CDS的克隆引物序列

Table 1 Primers sequence for the clone of CDS of gene erg4A,erg4B and erg4C

Gene	Primer sequence（5' - 3'）
erg4A	F:ATGGAGGACCAAGTCAAATC
erg4A	R:AAAAAGAGAAATTGCAGTAG
erg4B	F:ATGCCCTCCAAAAAGGACTCTC
erg4B	R:GTAAATTCCAGGAATGATGC
erg4C	F:ATGGATCGTCCCGGCTTCATT
erg4C	R:TTAGAAGACATAAGGAATGA

表2 用于构建GFP融合表达载体的引物序列

Table 2 Primer sequences for GFP fusion vector construction

Gene	Primer sequence（5' - 3'）
erg4A	CF:TCAACTCCATCACATCACAAGAGCTC ATGGAGG- ACC AAGTCAAATC
erg4A	CR:AGCTCCTCGCCCTTGCTCACTCTAGAAAAAAGAG- AA ATTGCAGTAG
erg4B	CF:TCAACTCCATCACATCACAA GAGCTC ATGCCCTC- CA AAAAGGACTC
erg4B	CR:AGCTCCTCGCCCTTGCTCAC TCTAGA GTAAATTC- CA GGAATGATGC
erg4C	NF:GCATGGACGAGCTGTACAAG GAGCTC ATGGATC- GTCCCGGCTTCAT
erg4C	NR:ATGGAGCTATTAAATCACTA TCTAGA TTAGAAG- ACA TACTATAGGA

表3 转化子验证所用引物序列

Table 3 Primer sequences for transformant validation

Gene	Primer sequence（5' - 3'）
C-CX	CX1:GGAGA CGTAT TTAGGTGCTA
C-CX	CX2:TGAACTTCA GGGTCAGCTT
N-CX	NX1:CGACAACCAC TACCTGAGCA
N-CX	NX2:TGAAGGGCGT ACTAGGGTTG
erg4A-GFP	F:ATGGAGGACCAAGTCAAATC
erg4A-GFP	R:AAAAAGAGAA ATTGCAGTAG
erg4B-GFP	F:ATGCCCTCCAAAAAGGACTC
erg4B-GFP	R:GTAAATTCCA GGAATGATGC
erg4C-GFP	F:ATGGATCGTC CCGGCTTCAT
erg4C-GFP	R:TTAGAAGACA TAAGGAATGA

表4 实时荧光定量PCR引物序列

Table 4 Primers for real-time fluorescent quantitative PCR

Gene	Primer sequence（5' - 3'）
erg4A	F:TCCACGAATGTTCGGAATCC
erg4A	R:AGCTCAAACCCAAGAGCATGA
erg4B	F:CGGCGCTTTATGATACCAATG
erg4B	R:GGGAAGGAAGACCTGCAAAAA
erg4C	F:GCATTCCCATCAATCAAAGCA
erg4C	R:CCGGGAAGAAGCACGTAACA
β-tubulin	F:CTCCAGCTCGAGCGTATGAAC
β-tubulin	R:GGCTCCAAATCGACGAGAAC

图1 erg4A、erg4B和erg4C的全长CDS克隆

Fig. 1 Full-length CDS cloning of erg4A,erg4B and erg4C

图2 erg4A、erg4B及erg4C的基因结构示意图

Fig. 2 Genetic structures of erg4A,erg4B and erg4C

图3 Erg4A、Erg4B和Erg4C的跨膜螺旋图中的红色矩形为跨膜螺旋结构,蓝色线段是位于膜内的结构,而红色线段是位于膜外的结构

Fig. 3 Transmembrane helices of Erg4A,Erg4B and Erg4C Red rectangle indicates the transmembrane,blue line segment indicates the structure inside the membrane,and the red line segment indicates the structure outside the membrane

图4 Erg4A、Erg4B和Erg4C蛋白亲疏水性图中的正值区域代表疏水氨基酸,负值区域代表亲水氨基酸

Fig. 4 Hydrophilicity/Hydrophobicity of Erg4A,Erg4B and Erg4C protein Positive areas represent the hydrophobic,the negative areas represent the hydrophilic

图5 GFP融合表达菌株PCR验证

Fig. 5 GFP fusion expression strain verified by PCR

图6 野生型与荧光标记菌株erg4A-GFP、erg4B-GFP及erg4C-GFP的菌落形态比较

Fig. 6 Comparison of colony morphologies of erg4A-GFP,erg4B-GFP and erg4C-GFP in wild type and fluor-esence labelled strains

图7 融合表达蛋白GFP的绿色荧光分布

Fig. 7 Distribution of green fluorescence of GFP fusion expression protein

图8 DAPI核染色与GFP的共定位

Fig. 8 Colocalization of DAPI nuclear staining with GFP

图9 内质网红色荧光探针染色与GFP的共定位

Fig. 9 Colocalization of endoplasmic reticulum red fluore-scent probe with GFP

图10 扩展青霉中erg4A、erg4B和erg4C在孢子阶段、孢子萌发阶段以及成熟菌丝阶段的相对表达量竖线表示标准误（±SE）. 不同字母代表显著性差异（P<0.05）,下同

Fig. 10 Relative expressions of erg4A,erg4B and erg4C in the spore,germinated spore and mycelium stages in P. expansum Vertical line indicates standard error（±SE）. Different letters refer to significant differences（P<0.05）. The same below

图11 扩展青霉中erg4A、erg4B和erg4C在CY固体和液体培养条件下的相对表达量

Fig. 11 Relative expressions of erg4A,erg4B and erg4C in P. expansum in Czapek yeast extract solid medium and Czapek yeast extract liquid medium

图12 扩展青霉中erg4A、erg4B和erg4C在黑暗和蓝光条件下的相对表达量

Fig. 12 Relative expressions of erg4A,erg4B and erg4C in P. expansum in dark and blue condition

参考文献 28

[1]	Sun C, Fu D, Lu HP, et al. Autoclaved yeast enhances the resistance against Penicillium expansum in postharvest pear fruit and its possible mechanisms of action[J]. Biological Control, 2018, 119:51-58. doi: 10.1016/j.biocontrol.2018.01.010 URL
[2]	Nierop Groot M, Abee T, van Bokhorst-van de Veen H. Inactivation of conidia from three Penicillium spp. isolated from fruit juices by conventional and alternative mild preservation technologies and disinfection treatments[J]. Food Microbiol, 2019, 81:108-114. doi: S0740-0020(17)31140-1 pmid: 30910081
[3]	Hu Z, He B, Ma L, et al. Recent advances in ergosterol biosynjournal and regulation mechanisms in Saccharomyces cerevisiae[J]. Indian J Microbiol, 2017, 57(3):270-277. doi: 10.1007/s12088-017-0657-1 URL
[4]	Sun L, Liao K. The effect of honokiol on ergosterol biosynjournal and vacuole function in Candida albicans[J]. Journal of Microbiol Biotechnology and biotechnology, 2020, 30(12):1835-1842.
[5]	Jin H, McCaffery JM, Grote E. Ergosterol promotes pheromone signaling and plasma membrane fusion in mating yeast[J]. J Cell Biol, 2008, 180(4):813-826. doi: 10.1083/jcb.200705076 URL
[6]	Song J, Zhai P, Zhang Y, et al. The Aspergillus fumigatus damage resistance protein family coordinately regulates ergosterol biosynjournal and azole susceptibility[J]. mBio, 2016, 7(1):e01919-e01915.
[7]	Alcazar-Fuoli L, Mellado E. Ergosterol biosynjournal in Aspergillus fumigatus:its relevance as an antifungal target and role in antifungal drug resistance[J]. Front Microbiol, 2012, 3:439. doi: 10.3389/fmicb.2012.00439 pmid: 23335918
[8]	Liu JF, Xia JJ, Nie KL, et al. Outline of the biosynjournal and regulation of ergosterol in yeast[J]. World J Microbiol Biotechnol, 2019, 35(7):98. doi: 10.1007/s11274-019-2673-2 URL
[9]	Yun Y, Yin D, Dawood DH, et al. Functional characterization of FgERG3 and FgERG5 associated with ergosterol biosynjournal, vegetative differentiation and virulence of Fusarium graminearum[J]. Fungal Genet Biol, 2014, 68:60-70. doi: 10.1016/j.fgb.2014.04.010 URL
[10]	Jordá T, Puig S. Regulation of ergosterol biosynjournal in Saccharomyces cerevisiae[J]. Genes, 2020, 11(7):795. doi: 10.3390/genes11070795 URL
[11]	Zweytick D, Hrastnik C, Kohlwein SD, et al. Biochemical characterization and subcellular localization of the sterol C-24(28)reductase, Erg4p, from the yeastSaccharomyces cerevisiae[J]. FEBS Lett, 2000, 470(1):83-87. pmid: 10722850
[12]	Feng W, Yang J, Xi Z, et al. Mutations and/or overexpressions of ERG4 and ERG11 genes in clinical azoles-resistant isolates of Candida albicans[J]. Microb Drug Resist, 2017, 23(5):563-570. doi: 10.1089/mdr.2016.0095 URL
[13]	Tiedje C, Holland DG, Just U, et al. Proteins involved in sterol synjournal interact with Ste20 and regulate cell polarity[J]. J Cell Sci, 2007, 120(pt 20):3613-3624. doi: 10.1242/jcs.009860 URL
[14]	Aguilar PS, Heiman MG, Walther TC, et al. Structure of sterol aliphatic chains affects yeast cell shape and cell fusion during mating[J]. PNAS, 2010, 107(9):4170-4175. doi: 10.1073/pnas.0914094107 pmid: 20150508
[15]	Bhattacharya S, Esquivel BD, White TC. Overexpression or deletion of ergosterol biosynjournal genes alters doubling time, response to stress agents, and drug susceptibility in Saccharomyces cerevisiae[J]. mBio, 2018, 9(4):e01291-e01218.
[16]	Venegas M, Barahona S, González AM, et al. Phenotypic analysis of mutants of ergosterol biosynjournal genes(ERG3 and ERG4)in the red yeast Xanthophyllomyces dendrorhous[J]. Front Microbiol, 2020, 11:1312. doi: 10.3389/fmicb.2020.01312 URL
[17]	Liu X, Jiang JH, Jiang JH, et al. Sterol C-24(28)reductase, encoded by FgERG4 gene, is important for ergosterol production, hyphal growth, sporulation and virulence in Fusarium graminearum[J]. Molecular plant pathology, 2013, 14(1):71-83. doi: 10.1111/mpp.2013.14.issue-1 URL
[18]	Long NB, Xu XL, Zeng QQ, et al. Erg4A and Erg4B are required for conidiation and azole resistance via regulation of ergosterol biosynjournal in Aspergillus fumigatus[J]. Appl Environ Microbiol, 2017, 83(4):e02924-e02916.
[19]	Li B, Zong Y, Du Z, et al. Genomic characterization reveals insights into patulin biosynjournal and pathogenicity in Penicillium species[J]. Mol Plant Microbe Interact, 2015, 28(6):635-647. doi: 10.1094/MPMI-12-14-0398-FI URL
[20]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4):402-408. pmid: 11846609
[21]	Tahir II, Department of Plant Breeding Swedish University of Agricultural Sciences Sweden, Nybom H, et al. Susceptibility to blue mold caused by Penicillium expansum in apple cultivars adapted to a cool climate[J]. Europ J Hort Sci, 2015, 80(3):117-127. doi: 10.17660/eJHS.2015/80.3.4 URL
[22]	刘馨. 小麦赤霉病菌麦角甾醇生物合成途径中关键基因的功能研究[D]. 杭州:浙江大学, 2012.
	Liu X. Functional analysis of genes involved in the ergosterol biosynthesis pathway in Fusarium graminearum[D]. Hangzhou:Zhejiang University, 2012.
[23]	Manchalu S, Mittal N, Spang A, et al. Local translation of yeast ERG4 mRNA at the endoplasmic Reticulum requires the brefeldin A resistance protein Bfr1[J]. RNA, 2019, 25(12):1661-1672. doi: 10.1261/rna.072017.119 URL
[24]	徐孝苓. 烟曲霉麦角固醇合成相关基因erg4的功能性研究[D]. 南京:南京师范大学, 2017.
	Xu XL. Functional analysis of ergosterol biosynthesis related gene erg4 in Aspergillus fumigatus[D]. Nanjing:Nanjing Normal University, 2017.
[25]	于忠洋, 姜洋, 翟明霞, 等. 桑黄麦角甾醇生物合成关键酶PlERG24基因克隆与表达分析[J]. 中草药, 2020, 51(22):5825-5832.
	Yu ZY, Jiang Y, Zhai MX, et al. Cloning and expression analysis of PlERG24 gene in ergosterol biosynjournal of Phellinus linteus[J]. Chin Tradit Herb Drugs, 2020, 51(22):5825-5832.
[26]	Rosenfeld E, Beauvoit B. Role of the non-respiratory pathways in the utilization of molecular oxygen by Saccharomyces cerevisiae[J]. Yeast, 2003, 20(13):1115-1144. pmid: 14558145
[27]	Tang Y, Zhu PK, Lu Z, et al. The photoreceptor components FaWC1 and FaWC2 of fusarium asiaticum cooperatively regulate light responses but play independent roles in virulence expression[J]. Microorganisms, 2020, 8(3):365. doi: 10.3390/microorganisms8030365 URL
[28]	Casas-Flores S, Rios-Momberg M, Bibbins M, et al. BLR-1 and BLR-2, key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride[J]. Microbiol Read Engl, 2004, 150(Pt 11):3561-3569. doi: 10.1099/mic.0.27346-0 URL