Bioinformatics,Subcellular Localization and Expression Analysis of erg4 in Penicillium expansum

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

Abstract

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

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.

Figures/Tables 16

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

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

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

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

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

Fig. 2 Genetic structures of erg4A,erg4B and erg4C

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

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

Fig. 5 GFP fusion expression strain verified by PCR

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

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

Fig. 8 Colocalization of DAPI nuclear staining with GFP

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

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

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

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

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