- Email: [email protected]
Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis
Accepted Manuscript Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis Junjun Shang, Ruiheng Yang, Lihua Tang, Yán Li, Yan Li, Wenjun Mao, Ming Gong, Ying Wang, Yoichi Honda, Dapeng Bao PII:
S1340-3540(18)30246-8
DOI:
https://doi.org/10.1016/j.myc.2019.06.004
Reference:
MYC 453
To appear in:
Mycoscience
Received Date: 29 September 2018 Revised Date:
6 June 2019
Accepted Date: 13 June 2019
Please cite this article as: Shang J, Yang R, Tang L, Li Yá, Li Y, Mao W, Gong M, Wang Y, Honda Y, Bao D, Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis, Mycoscience (2019), doi: https://doi.org/10.1016/j.myc.2019.06.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis
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Junjun Shang a,1, Ruiheng Yang a,1, Lihua Tang a,1, Yán Li a, Yan Li a, Wenjun Mao a, Ming Gong a, Ying Wang a, Yoichi Honda b, Dapeng Bao a,*
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Affiliation:
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Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, PR China Address: 1000 Jingqi road, Fengxian District, Shanghai, PR China, 201403
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Address: Kitashirakawa-oiwake-cho, Sakyoku-ku, Kyoto 606-8502, Japan
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These authors contributed equally to this study
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*
Corresponding author:
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Dapeng Bao: telephone: 0086-21-62200794; fax: 0086-21-62201337; Email: [email protected]; address: 1000 Jingqi road, Fengxian District, Shanghai, PR China, 201403
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Declarations of interest: none
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Laboratory of Forest Biochemistry, Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University
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Abstract: Transcriptional analysis showed that Pegpd1 was constitutively and highly expressed in various developmental stages of P. eryngii. The expression level of Pegpd2 was much lower than Pegpd1. The promoter of Pegpd1 contained the conserved cis-regulatory elements and could be used to drive heterologous gene expression in genetic transformation of P. eryngii.
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key words: Glyceraldehyde-3-phosphate dehydrogenase (GPD), transcriptional analysis, promoter
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ACCEPTED MANUSCRIPT Glyceraldehyde-3-phosphate dehydrogenase (GPD), one of the key enzymes in the glycolytic pathway, catalyzes the NADH-dependent conversion of dihydroxyacetone phosphate to glycerol-3 phosphate (Knudsen et al., 2015). gpd genes were identified widely in human, animals, plants and fungi. (Ercolani et al., 1988; Fort et al., 1985; Musti et al., 1983; Shih et al., 1991). gpd gene family of different species might contain one to several members. Comparison between GPDs revealed that the GPD proteins were highly conserved and contained the NAD+binding domain, the C-terminal catalytic domain and the main conserved amino acid residues, which functioned as the binding site of the enzyme in the catalytic region (Fort et al., 1985; Musti et al., 1983; Tasaki et al., 2014).
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Pleurotus eryngii is an important edible and medicinal mushroom which is commercially cultivated in world wide area. Pleurotus ostreatus is a closely related species of P. eryngii and contains two gpds. In this study, the DNA sequences of Pogpd1 and Pogpd2 from P. ostreatus (Tasaki et al., 2014) were used to search the genome of P. eryngii monokaryon strain ‘183’ (Yang et al., 2016). The BLAST (Basic Local Alignment Search Tool) analysis (Altschul et al., 1990) showed that there are two gpd genes in P. eryngii . We named them Pegpd1 and Pegpd2. Pegpd1 (99.4% identity with PoGPD1 at the amino acid level) was located between bp 55309 and 53833 on the scaffold107 (GenBank accession No. MAZY01000107) and Pegpd2 (98.5% identity with PoGPD2 at the amino acid level) was located between bp 20984 and 22447 on the scaffold 128 (Genbank accession No. MAZY01000128). The two Pegpds from P.eryngii also showed extensive homology to each other. The alignment of their coding sequences was shown in Supplementary Fig. S1.
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We compared the amino acid sequences of PeGPDs with the published GPDs from basidiomycetes other than P. ostreatus. The PeGPDs were highly similar to the GPDs from P. sajor-caju (92.5–96.4% identity) (Jeong et al., 2000), Hypsizygus marmoreus (81.1–82.2% identity) (Zhang et al., 2014), Lentinus edodes (79.5–79.8% identity) (Hirano et al., 1999) and Agaricus bisporus (66.8–76.0% identity) (Harmsen et al., 1992). An evolutionary tree based on the GPD amino acid sequences was performed using the neighbor joining method (Saitou and Nei, 1987) in MEGA6 (Tamura et al., 2013). Phylogenetic analysis showed that the relationship between the GPDs reflected the evolutionary lineages of the species encoding these proteins (Fig. 1).
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In many organisms, gpds are highly expressed genes, for example, GPD mRNA accounts for 2–5% of total poly (A)+ RNA in Saccharomyces cerevisiae (Holland and Holland, 1978). There are some species that have two or more differentially expressed gpd genes. Various rat adult tissues express only one major mRNA species from gpd multigenic family (Fort et al., 1985). In A. bisporus, the gpd2 gene was strongly expressed in both mycelium and fruit bodies, while no transcripts of gpd1 had been found (Harmsen et al., 1992). In Mucor circinelloides,
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transcripts of gpd1 could be detected during vegetative growth, while no transcripts from gpd2 and gpd3 could be detected (Wolff and Arnau, 2002). To reveal the expression patterns of Pegpd1 and Pegpd2, we investigated the reads of these two genes in the RNA sequencing datasets from the P. eryngii strain ‘Xinhan’. These datasets included the mRNA transcriptome data from the tissue samples collected through P. eryngii fruitbody development. Although Pegpd1 is very similar to Pegpd2 at the deduced amino acid level, there are 145 SNPs (single nucleotide polymorphisms) between them in the 1011 nt (nucleotides) of the coding sequences. Using these SNPs, we distinguished the reads from Pegpd1 and Pegpd2 in the transcriptome data (Supplementary Table S1). The unit of RPKM (Reads Per Kilobase per Million mapped reads) was used to compare the relative mRNA expression levels. The analysis showed that Pegpd1 was constitutively and highly expressed in various developmental stages of P. eryngii, including mycelial , primordial, young fruit body and mature fruit body stages. The expression level of Pegpd2 is much lower than Pegpd1 in all stages (Fig. 2). The expression pattern of the Pegpds is very different with the Pogpds in P. ostreatus, in which transcript levels of Pogpd2 were high in the mycelial and mature fruit body stage (Tasaki et al., 2014).
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The 1-kb region from the start codon ATG of Pegpd1 were investigated for the conserved elements in eukaryotic promoters. The transcription start point was predicted 61 nt upstream from the first ATG (http://www.fruitfly.org/seq_tools/promoter.html). Three CAAT boxes were located at -380 nt, -342 nt and -248 nt from the transcription start point. The TATA box was located at -29 nt from the transcription start point (Fig. 3). The results showed that the 1kb region has the conserved elements of a typical eukaryotic promoter.
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Due to the high expression of gpds, the gpd promoter sequences had been used in vectors for heterologous gene expression in basidiomycetes such as L. edodes (Hirano et al., 2000), P. ostreatus (Irie et al., 2001), A. bisporus (Burns et al., 2005) and Coprinopsis cinerea (Kilaru et al., 2006). In a species containing several gpds, it is crucial to choose the promoter from the right copy of gpds, otherwise the promoter might not work in some developmental stages (Tasaki et al., 2014) or environmental conditions (Knudsen et al., 2015), or even might not work at all (Harmsen et al., 1992; Wolff and Arnau, 2002). Combing the results from the transcriptional analysis and the sequence analysis in our lab, we concluded that Pegpd1 was constitutively and highly expressed during all the developmental stages and Pegpd1 promoter could be used to drive heterologous gene expression in genetic transformation of P. eryngii. The promoter of Pegpd1 was used to drive Cas9 gene expression in P. eryngii. The plasmid containing the Pegpd1-promoter::Cas9 cassette was introduced into the wild type Pleurotus eryngii strain DX8 by PEG-mediated transformation. PCR analysis was used to detect the integration of Cas9 gene in the transfromants. As shown in the Supplementary Fig. S2, the Cas9 fragment could be amplified from the genomic DNA of the transformant 1810 (lane 5), but could not be amplified from the genomic DNA
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ACCEPTED MANUSCRIPT of DX8 (lane 4, as a negative control), which demonstrated that Cas9 gene was integrated into the genome of the transformant 1810. Then RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis was performed to check the expression of Cas9 gene from the transfromant 1810. The Cas9 fragment could be amplified from the cDNA of the transformant 1810 (lane 3), but could not be amplified from the cDNA of DX8 (lane 2, as a negative control), which demonstrated that the Cas9 gene driven by the Pegpd1 promoter was successfully expressed in the transformant 1810.
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This study was supported by National Natural Science Foundation of China (Grant No. 31660296); the Shanghai Science and Technology Committee (STCSM) Applied Technology Development Program (Grant No. 17391900400); the start-up funding from Shanghai Agriculture Academies of Sciences to Junjun Shang.
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References:
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Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riquelme, M., Elliott, T.J., Challen, M.P., Bailey, A., and Foster, G.D. (2005). Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genetics & Biology 42, 191–199. Ercolani, L., Florence, B., Denaro, M., and Alexander, M. (1988). Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. The Journal of Biological Chemistry 263, 15335–15341. Fort, P., Marty, L., Piechaczyk, M., el Sabrouty, S., Dani, C., Jeanteur, P., and Blanchard, J.M. (1985). Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Research 13, 1431– 1442. Harmsen, M.C., Schuren, F.H.J., Moukha, S.M., Zuilen, C.M.V., Punt, P.J., and Wessels, J.G.H. (1992). Sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase genes from the basidiomycetes Schizophyllum commune, Phanerochaete chrysosporium and Agaricus bisporus. Current Genetics 22, 447–454. Hirano, T., Sato, T., Okawa, K., Kanda, K., Yaegashi, K., and Enei, H. (1999). Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Lentinus edodes. Bioscience, Biotechnology, and Biochemistry 63, 1223–1227. Hirano, T., Sato, T., Yaegashi, K., and Enei, H. (2000). Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance. Molecular and General Genetics 263, 1047–1052. Holland, M.J., and Holland, J.P. (1978). Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry 17, 4900–4907. Irie, T., Honda, Y., Watanabe, T., and Kuwahara, M. (2001). Homologous expression of recombinant manganese peroxidase genes in ligninolytic fungus Pleurotus ostreatus. Applied Microbiology & Biotechnology 55, 566.
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Jeong, M.J., Park, S.C., Kwon, H.B., and Byun, M.O. (2000). Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase. Biochemical & Biophysical Research Communications 278, 192–196. Kilaru, S., Hoegger, P.J., Majcherczyk, A., Burns, C., Shishido, K., Bailey, A., Foster, G.D., and Kües, U. (2006). Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Applied Microbiology & Biotechnology 71, 200–210. Knudsen, J.D., Johanson, T., Eliasson Lantz, A., and Carlquist, M. (2015). Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae. Biotechnology Reports (Amsterdam, Netherlands) 7, 107–119. Musti, A.M., Zehner, Z., Bostian, K.A., Paterson, B.M., and Kramer, R.A. (1983). Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene. Gene 25, 133–143. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425. Shih, M.C., Heinrich, P., and Goodman, H.M. (1991). Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana. Gene 104, 133–138. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30, 2725–2729. Tasaki, Y., Sato, R., Toyama, S., Kasahara, K., Ona, Y., and Sugawara, M. (2014). Cloning of glyceraldehyde-3-phosphate dehydrogenase genes from the basidiomycete mushroom Pleurotus ostreatus and analysis of their expression during fruit-body development. Mycoscience 55, 280– 288. Wolff, A.M., and Arnau, J. (2002). Cloning of Glyceraldehyde-3-phosphate DehydrogenaseEncoding Genes in Mucor circinelloides (Syn. racemosus) and Use of the gpd1 Promoter for Recombinant Protein Production. Fungal Genetics and Biology 35, 21–29. Yang, R.H., Li, Y., Wáng, Y., Wan, J.N., Zhou, C.L., Wāng, Y., Gao, Y.N., Mao, W.J., Tang, L.H., and Gong, M. (2016). The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay. Journal of Biotechnology 239, 65–67. Zhang, J., Shi, L., Chen, H., Sun, Y., Zhao, M., Ren, A., Chen, M., Wang, H., and Feng, Z. (2014). An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus. Microbiological Research 169, 741.
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Fig. 1. Phylogenetic analysis of the published GPD amino acid sequences from basidiomycetes. The names of the species were followed by the Genbank accession numbers of the GPDs. Arrows indicated the GPDs from Pleurotus eryngii. Phylogenetic analysis were performed using the neighbor joining method in MEGA6.
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Fig. 2. Comparison of the relative expression of Pegpd1 and Pegpd2. Pegpd1 was constitutive and highly expressed in various developmental stages of Pleurotus eryngii, including mycelial , primordial, young fruit body and mature fruit body stages. M: Mycelia. M10: Mycelia collected 10 d after inoculation. M20: Mycelia 5
ACCEPTED MANUSCRIPT collected 20 d after inoculation. S: Mycelia collected in scratch period. P: Primordia. F1: Young fruit bodies. F2: Mature fruit bodies.
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Fig. 3. The 1-kb promoter region of Pegpd1 upstream from the translation start codon ATG. The predicted transcription start point was indicated by an arrow. Single and double under lines indicated the CAAT boxes and TATA box, respectively.
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Highlights: •
Pleurotus eryngii contained two gpd genes that were similar to other gpds
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Pegpd1 was constitutively and highly expressed in various
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developmental stages
Pegpd1 promoter could be used to drive gene expression in genetic
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transformation
S1340-3540(18)30246-8
DOI:
https://doi.org/10.1016/j.myc.2019.06.004
Reference:
MYC 453
To appear in:
Mycoscience
Received Date: 29 September 2018 Revised Date:
6 June 2019
Accepted Date: 13 June 2019
Please cite this article as: Shang J, Yang R, Tang L, Li Yá, Li Y, Mao W, Gong M, Wang Y, Honda Y, Bao D, Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis, Mycoscience (2019), doi: https://doi.org/10.1016/j.myc.2019.06.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1 2
Differential expression of two gpd genes in the cultivated mushroom Pleurotus eryngii using RNA sequencing analysis
3 4 5
Junjun Shang a,1, Ruiheng Yang a,1, Lihua Tang a,1, Yán Li a, Yan Li a, Wenjun Mao a, Ming Gong a, Ying Wang a, Yoichi Honda b, Dapeng Bao a,*
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Affiliation:
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Shanghai Key Laboratory of Agricultural Genetics and Breeding, Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Shanghai, PR China Address: 1000 Jingqi road, Fengxian District, Shanghai, PR China, 201403
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b
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Address: Kitashirakawa-oiwake-cho, Sakyoku-ku, Kyoto 606-8502, Japan
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These authors contributed equally to this study
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*
Corresponding author:
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Dapeng Bao: telephone: 0086-21-62200794; fax: 0086-21-62201337; Email: [email protected]; address: 1000 Jingqi road, Fengxian District, Shanghai, PR China, 201403
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Declarations of interest: none
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Laboratory of Forest Biochemistry, Division of Environmental Science and Technology, Graduate School of Agriculture, Kyoto University
EP
Abstract: Transcriptional analysis showed that Pegpd1 was constitutively and highly expressed in various developmental stages of P. eryngii. The expression level of Pegpd2 was much lower than Pegpd1. The promoter of Pegpd1 contained the conserved cis-regulatory elements and could be used to drive heterologous gene expression in genetic transformation of P. eryngii.
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key words: Glyceraldehyde-3-phosphate dehydrogenase (GPD), transcriptional analysis, promoter
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ACCEPTED MANUSCRIPT Glyceraldehyde-3-phosphate dehydrogenase (GPD), one of the key enzymes in the glycolytic pathway, catalyzes the NADH-dependent conversion of dihydroxyacetone phosphate to glycerol-3 phosphate (Knudsen et al., 2015). gpd genes were identified widely in human, animals, plants and fungi. (Ercolani et al., 1988; Fort et al., 1985; Musti et al., 1983; Shih et al., 1991). gpd gene family of different species might contain one to several members. Comparison between GPDs revealed that the GPD proteins were highly conserved and contained the NAD+binding domain, the C-terminal catalytic domain and the main conserved amino acid residues, which functioned as the binding site of the enzyme in the catalytic region (Fort et al., 1985; Musti et al., 1983; Tasaki et al., 2014).
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Pleurotus eryngii is an important edible and medicinal mushroom which is commercially cultivated in world wide area. Pleurotus ostreatus is a closely related species of P. eryngii and contains two gpds. In this study, the DNA sequences of Pogpd1 and Pogpd2 from P. ostreatus (Tasaki et al., 2014) were used to search the genome of P. eryngii monokaryon strain ‘183’ (Yang et al., 2016). The BLAST (Basic Local Alignment Search Tool) analysis (Altschul et al., 1990) showed that there are two gpd genes in P. eryngii . We named them Pegpd1 and Pegpd2. Pegpd1 (99.4% identity with PoGPD1 at the amino acid level) was located between bp 55309 and 53833 on the scaffold107 (GenBank accession No. MAZY01000107) and Pegpd2 (98.5% identity with PoGPD2 at the amino acid level) was located between bp 20984 and 22447 on the scaffold 128 (Genbank accession No. MAZY01000128). The two Pegpds from P.eryngii also showed extensive homology to each other. The alignment of their coding sequences was shown in Supplementary Fig. S1.
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We compared the amino acid sequences of PeGPDs with the published GPDs from basidiomycetes other than P. ostreatus. The PeGPDs were highly similar to the GPDs from P. sajor-caju (92.5–96.4% identity) (Jeong et al., 2000), Hypsizygus marmoreus (81.1–82.2% identity) (Zhang et al., 2014), Lentinus edodes (79.5–79.8% identity) (Hirano et al., 1999) and Agaricus bisporus (66.8–76.0% identity) (Harmsen et al., 1992). An evolutionary tree based on the GPD amino acid sequences was performed using the neighbor joining method (Saitou and Nei, 1987) in MEGA6 (Tamura et al., 2013). Phylogenetic analysis showed that the relationship between the GPDs reflected the evolutionary lineages of the species encoding these proteins (Fig. 1).
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In many organisms, gpds are highly expressed genes, for example, GPD mRNA accounts for 2–5% of total poly (A)+ RNA in Saccharomyces cerevisiae (Holland and Holland, 1978). There are some species that have two or more differentially expressed gpd genes. Various rat adult tissues express only one major mRNA species from gpd multigenic family (Fort et al., 1985). In A. bisporus, the gpd2 gene was strongly expressed in both mycelium and fruit bodies, while no transcripts of gpd1 had been found (Harmsen et al., 1992). In Mucor circinelloides,
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transcripts of gpd1 could be detected during vegetative growth, while no transcripts from gpd2 and gpd3 could be detected (Wolff and Arnau, 2002). To reveal the expression patterns of Pegpd1 and Pegpd2, we investigated the reads of these two genes in the RNA sequencing datasets from the P. eryngii strain ‘Xinhan’. These datasets included the mRNA transcriptome data from the tissue samples collected through P. eryngii fruitbody development. Although Pegpd1 is very similar to Pegpd2 at the deduced amino acid level, there are 145 SNPs (single nucleotide polymorphisms) between them in the 1011 nt (nucleotides) of the coding sequences. Using these SNPs, we distinguished the reads from Pegpd1 and Pegpd2 in the transcriptome data (Supplementary Table S1). The unit of RPKM (Reads Per Kilobase per Million mapped reads) was used to compare the relative mRNA expression levels. The analysis showed that Pegpd1 was constitutively and highly expressed in various developmental stages of P. eryngii, including mycelial , primordial, young fruit body and mature fruit body stages. The expression level of Pegpd2 is much lower than Pegpd1 in all stages (Fig. 2). The expression pattern of the Pegpds is very different with the Pogpds in P. ostreatus, in which transcript levels of Pogpd2 were high in the mycelial and mature fruit body stage (Tasaki et al., 2014).
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The 1-kb region from the start codon ATG of Pegpd1 were investigated for the conserved elements in eukaryotic promoters. The transcription start point was predicted 61 nt upstream from the first ATG (http://www.fruitfly.org/seq_tools/promoter.html). Three CAAT boxes were located at -380 nt, -342 nt and -248 nt from the transcription start point. The TATA box was located at -29 nt from the transcription start point (Fig. 3). The results showed that the 1kb region has the conserved elements of a typical eukaryotic promoter.
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Due to the high expression of gpds, the gpd promoter sequences had been used in vectors for heterologous gene expression in basidiomycetes such as L. edodes (Hirano et al., 2000), P. ostreatus (Irie et al., 2001), A. bisporus (Burns et al., 2005) and Coprinopsis cinerea (Kilaru et al., 2006). In a species containing several gpds, it is crucial to choose the promoter from the right copy of gpds, otherwise the promoter might not work in some developmental stages (Tasaki et al., 2014) or environmental conditions (Knudsen et al., 2015), or even might not work at all (Harmsen et al., 1992; Wolff and Arnau, 2002). Combing the results from the transcriptional analysis and the sequence analysis in our lab, we concluded that Pegpd1 was constitutively and highly expressed during all the developmental stages and Pegpd1 promoter could be used to drive heterologous gene expression in genetic transformation of P. eryngii. The promoter of Pegpd1 was used to drive Cas9 gene expression in P. eryngii. The plasmid containing the Pegpd1-promoter::Cas9 cassette was introduced into the wild type Pleurotus eryngii strain DX8 by PEG-mediated transformation. PCR analysis was used to detect the integration of Cas9 gene in the transfromants. As shown in the Supplementary Fig. S2, the Cas9 fragment could be amplified from the genomic DNA of the transformant 1810 (lane 5), but could not be amplified from the genomic DNA
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ACCEPTED MANUSCRIPT of DX8 (lane 4, as a negative control), which demonstrated that Cas9 gene was integrated into the genome of the transformant 1810. Then RT-PCR (Reverse Transcription-Polymerase Chain Reaction) analysis was performed to check the expression of Cas9 gene from the transfromant 1810. The Cas9 fragment could be amplified from the cDNA of the transformant 1810 (lane 3), but could not be amplified from the cDNA of DX8 (lane 2, as a negative control), which demonstrated that the Cas9 gene driven by the Pegpd1 promoter was successfully expressed in the transformant 1810.
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This study was supported by National Natural Science Foundation of China (Grant No. 31660296); the Shanghai Science and Technology Committee (STCSM) Applied Technology Development Program (Grant No. 17391900400); the start-up funding from Shanghai Agriculture Academies of Sciences to Junjun Shang.
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References:
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Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403–410. Burns, C., Gregory, K.E., Kirby, M., Cheung, M.K., Riquelme, M., Elliott, T.J., Challen, M.P., Bailey, A., and Foster, G.D. (2005). Efficient GFP expression in the mushrooms Agaricus bisporus and Coprinus cinereus requires introns. Fungal Genetics & Biology 42, 191–199. Ercolani, L., Florence, B., Denaro, M., and Alexander, M. (1988). Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. The Journal of Biological Chemistry 263, 15335–15341. Fort, P., Marty, L., Piechaczyk, M., el Sabrouty, S., Dani, C., Jeanteur, P., and Blanchard, J.M. (1985). Various rat adult tissues express only one major mRNA species from the glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucleic Acids Research 13, 1431– 1442. Harmsen, M.C., Schuren, F.H.J., Moukha, S.M., Zuilen, C.M.V., Punt, P.J., and Wessels, J.G.H. (1992). Sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase genes from the basidiomycetes Schizophyllum commune, Phanerochaete chrysosporium and Agaricus bisporus. Current Genetics 22, 447–454. Hirano, T., Sato, T., Okawa, K., Kanda, K., Yaegashi, K., and Enei, H. (1999). Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Lentinus edodes. Bioscience, Biotechnology, and Biochemistry 63, 1223–1227. Hirano, T., Sato, T., Yaegashi, K., and Enei, H. (2000). Efficient transformation of the edible basidiomycete Lentinus edodes with a vector using a glyceraldehyde-3-phosphate dehydrogenase promoter to hygromycin B resistance. Molecular and General Genetics 263, 1047–1052. Holland, M.J., and Holland, J.P. (1978). Isolation and identification of yeast messenger ribonucleic acids coding for enolase, glyceraldehyde-3-phosphate dehydrogenase, and phosphoglycerate kinase. Biochemistry 17, 4900–4907. Irie, T., Honda, Y., Watanabe, T., and Kuwahara, M. (2001). Homologous expression of recombinant manganese peroxidase genes in ligninolytic fungus Pleurotus ostreatus. Applied Microbiology & Biotechnology 55, 566.
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Jeong, M.J., Park, S.C., Kwon, H.B., and Byun, M.O. (2000). Isolation and characterization of the gene encoding glyceraldehyde-3-phosphate dehydrogenase. Biochemical & Biophysical Research Communications 278, 192–196. Kilaru, S., Hoegger, P.J., Majcherczyk, A., Burns, C., Shishido, K., Bailey, A., Foster, G.D., and Kües, U. (2006). Expression of laccase gene lcc1 in Coprinopsis cinerea under control of various basidiomycetous promoters. Applied Microbiology & Biotechnology 71, 200–210. Knudsen, J.D., Johanson, T., Eliasson Lantz, A., and Carlquist, M. (2015). Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae. Biotechnology Reports (Amsterdam, Netherlands) 7, 107–119. Musti, A.M., Zehner, Z., Bostian, K.A., Paterson, B.M., and Kramer, R.A. (1983). Transcriptional mapping of two yeast genes coding for glyceraldehyde 3-phosphate dehydrogenase isolated by sequence homology with the chicken gene. Gene 25, 133–143. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4, 406–425. Shih, M.C., Heinrich, P., and Goodman, H.M. (1991). Cloning and chromosomal mapping of nuclear genes encoding chloroplast and cytosolic glyceraldehyde-3-phosphate-dehydrogenase from Arabidopsis thaliana. Gene 104, 133–138. Tamura, K., Stecher, G., Peterson, D., Filipski, A., and Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30, 2725–2729. Tasaki, Y., Sato, R., Toyama, S., Kasahara, K., Ona, Y., and Sugawara, M. (2014). Cloning of glyceraldehyde-3-phosphate dehydrogenase genes from the basidiomycete mushroom Pleurotus ostreatus and analysis of their expression during fruit-body development. Mycoscience 55, 280– 288. Wolff, A.M., and Arnau, J. (2002). Cloning of Glyceraldehyde-3-phosphate DehydrogenaseEncoding Genes in Mucor circinelloides (Syn. racemosus) and Use of the gpd1 Promoter for Recombinant Protein Production. Fungal Genetics and Biology 35, 21–29. Yang, R.H., Li, Y., Wáng, Y., Wan, J.N., Zhou, C.L., Wāng, Y., Gao, Y.N., Mao, W.J., Tang, L.H., and Gong, M. (2016). The genome of Pleurotus eryngii provides insights into the mechanisms of wood decay. Journal of Biotechnology 239, 65–67. Zhang, J., Shi, L., Chen, H., Sun, Y., Zhao, M., Ren, A., Chen, M., Wang, H., and Feng, Z. (2014). An efficient Agrobacterium-mediated transformation method for the edible mushroom Hypsizygus marmoreus. Microbiological Research 169, 741.
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Fig. 1. Phylogenetic analysis of the published GPD amino acid sequences from basidiomycetes. The names of the species were followed by the Genbank accession numbers of the GPDs. Arrows indicated the GPDs from Pleurotus eryngii. Phylogenetic analysis were performed using the neighbor joining method in MEGA6.
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Fig. 2. Comparison of the relative expression of Pegpd1 and Pegpd2. Pegpd1 was constitutive and highly expressed in various developmental stages of Pleurotus eryngii, including mycelial , primordial, young fruit body and mature fruit body stages. M: Mycelia. M10: Mycelia collected 10 d after inoculation. M20: Mycelia 5
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Fig. 3. The 1-kb promoter region of Pegpd1 upstream from the translation start codon ATG. The predicted transcription start point was indicated by an arrow. Single and double under lines indicated the CAAT boxes and TATA box, respectively.
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Pleurotus eryngii contained two gpd genes that were similar to other gpds
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Pegpd1 was constitutively and highly expressed in various
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Pegpd1 promoter could be used to drive gene expression in genetic
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