International Journal of
Molecular Sciences Article
The Sequence Characteristics and Expression Models Reveal Superoxide Dismutase Involved in Cold Response and Fruiting Body Development in Volvariella volvacea Jun-Jie Yan 1 , Lei Zhang 1 , Rui-Qing Wang 1,2 , Bin Xie 1 , Xiao Li 1 , Ren-Liang Chen 1 , Li-Xian Guo 1 and Bao-Gui Xie 1, * Received: 13 November 2015; Accepted: 21 December 2015; Published: 14 January 2016 Academic Editor: Patrick C.Y. Woo 1
2
*
Mycological Research Center, College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
[email protected] (J.-J.Y.);
[email protected] (L.Z.);
[email protected] (R.-Q.W.);
[email protected] (B.X.);
[email protected] (X.L.);
[email protected] (R.-L.C.);
[email protected] (L.-X.G.) College of Food Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China Correspondence:
[email protected]; Tel./Fax: +86-591-8378-9277
Abstract: As the first defence for cells to counteract the toxicity of active oxygen, superoxide dismutase (SOD) plays an important role in the response of living organisms to stress and cell differentiation. One extracellular Cu-ZnSOD (ecCu-ZnSOD), and two MnSODs, were identified based on the Volvariella volvacea genome sequence. All three genes have complicated alternative splicing modes during transcription; only when the fourth intron is retained can the Vv_Cu-Znsod1 gene be translated into a protein sequence with SOD functional domains. The expression levels of the three sod genes in the pilei are higher than in the stipe. The Vv_Cu-Znsod1 and the Vv_Mnsod2 are co-expressed in different developmental stages of the fruiting body, with the highest level of expression in the pilei of the egg stage, and they show a significant, positive correlation with the efficiency of karyogamy, indicating the potential role of these two genes during karyogamy. The expression of the ecCu-Znsod and two Vv_Mnsod genes showed a significant up-regulated when treated by cold stress for one hour; however, the lack of the intracellular Cu-ZnSOD encoding gene (icCu-Znsod) and the special locus of the ecCu-Znsod gene initiation codon suggested a possible reason for the autolysis phenomenon of V. volvacea in cold conditions. Keywords: straw mushroom; alternative splicing modes; gene differential expression; cold stress; fruiting-body development
1. Introduction Superoxide dismutases (SOD, EC 1.15.1.1) constitute a family of important enzymes that scavenge oxygen free radicals. As the first defence in cells to counteract the toxicity of active oxygen, these enzymes exist in various aerobic organisms [1,2]. SODs are classified into four groups according to their binding metal ions: Fe, Ni, Mn, and Cu-Zn. MnSOD and Cu-ZnSOD are found in eukaryotes and in most prokaryotes. FeSOD, however, is found mostly in prokaryotes and in a few plants, and NiSOD is only found in Streptomyces and cyanobacteria [3–5]. Many studies have indicated that although Cu-ZnSOD and MnSOD have different crystal structures, metal cofactors, catalytic mechanisms and amino acid sequences, they play similar and important roles in biological processes, including scavenging of reactive oxygen species (ROS), environmental stress responses, cell differentiation and ageing [6–13]. Int. J. Mol. Sci. 2016, 17, 34; doi:10.3390/ijms17010034
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Volvariella volvacea is an important edible mushroom cultivated seasonally in tropical and subtropical areas, and the growth of the fruiting body is susceptible to environmental stress [14–16]. It is very sensitive to cold conditions that cause deterioration or even autolysis of hyphae, and have a negative impact on mushroom production and preservation [17,18]. Li et al. [19] determined the SOD enzyme activity and developed isozyme electrophoretograms for the different developmental stages of the fruiting body of V. volvacea. They found that the SOD enzyme activity increases gradually as the fruiting matures, of the Int.body J. Mol. Sci. 2016, 17, 34 and the number of isozyme bands is the smallest when the 2activity of 13 enzyme is the highest. Li et al. [20] also found differences among SOD isozyme patterns in V. volvacea Volvariella volvacea is an important edible mushroom cultivated seasonally in tropical and mycelia atsubtropical differentareas, ages,and inthe different growth periods of the fruiting body and in the fruiting body growth of the fruiting body is susceptible to environmental stress [14–16]. stored at aItlow temperature for different time periods. All oforthese results show thatand SOD is involved is very sensitive to cold conditions that cause deterioration even autolysis of hyphae, have a negative impact on mushroom production and preservation [17,18]. Li et al. [19] determined the in regulating biological processes, including mycelial aging, fruiting body development and response SOD Although, enzyme activity forstudied the different developmental to cold stress. theand soddeveloped genes ofisozyme severalelectrophoretograms mushrooms were [21,22], there has been no stages of the fruiting body of V. volvacea. They found that the SOD enzyme activity increases gradually as report of the identification of SOD encoding genes or of gene regulation at the transcriptional level in the fruiting body matures, and the number of isozyme bands is the smallest when the activity of the V. volvacea.enzyme Underis laboratory sequenced the genome and transcriptome of V. volvacea the highest. Liconditions, et al. [20] also we found differences among SOD isozyme patterns in V. volvacea mycelia at different ages, inSOD different growth periods of the fruiting body and inmethod. the fruitingBy body stored and identified genes encoding using a bioinformatics annotation analyzing the sod at a low temperature for different time periods. All of these results show that SOD is involved in regulating genetic structure and detecting its expression in different samples, we initiated studies of the biological biological processes, including mycelial aging, fruiting body development and response to cold stress. function ofAlthough, SOD atthe thesod gene level and at the transcriptional level in V. volvacea. genes of several mushrooms were studied [21,22], there has been no report of the identification of SOD encoding genes or of gene regulation at the transcriptional level in V. volvacea. Under
2. Resultslaboratory conditions, we sequenced the genome and transcriptome of V. volvacea and identified genes encoding SOD using a bioinformatics annotation method. By analyzing the sod genetic structure and
2.1. Annotation anditsSequence of SODstudies Encoding Genes function of SOD at the detecting expressionAccuracy in different Verification samples, we initiated of the biological gene level and at the transcriptional level in V. volvacea.
The protein-protein BLAST (BLASTP) results showed that the V. volvacea genome contained one 2. Results Cu-ZnSOD (encoding gene prediction No. GME1978) and two MnSODs (encoding gene prediction Nos. GME5987 and GME8899), but no FeSOD or NiSOD. 2.1. Annotation and Sequence Accuracy Verification of SOD Encoding Genes The DNA sequences encoding the three SODs were obtained from the genome based on the gene The protein-protein BLAST (BLASTP) results showed that the V. volvacea genome contained one prediction numbers and gene models were annotated as described in the supplementary methods Cu-ZnSOD (encoding gene prediction No. GME1978) and two MnSODs (encoding gene prediction (S1). The Nos. results suggested that the oforGME1978 (Vv_Cu-Znsod1), GME5987 (Vv-Mnsod1), GME5987 and GME8899), butlengths no FeSOD NiSOD. and GME8899The (Vv-Mnsod2) were 1255 bps,obtained and 1589 respectively. DNA sequences encoding thebps, three 1110 SODs were from bps, the genome based on theThe geneelongated numbersby and gene models were annotated as described in the supplementary methods sequencesprediction were mapped the reads obtained by genome sequencing, and the results are shown in (S1). The results suggested that the lengths of GME1978 (Vv_Cu-Znsod1), GME5987 (Vv-Mnsod1), and supplementary Figure S1. The base pairs of the full-length gene sequences had at least nine reads GME8899 (Vv-Mnsod2) were 1255 bps, 1110 bps, and 1589 bps, respectively. The elongated sequences mapped. The using the Sanger method confirmed the three genein sequences weresequencing mapped by results the reads obtained by genome sequencing, and that the results are sod shown supplementary S1. for The further base pairsstudy. of the full-length gene sequences had at least nine reads were correct and could Figure be used mapped. The sequencing results using the Sanger method confirmed that the three sod gene were correct and could be used Analysis for further study. 2.2. Geneticsequences Structure and Alternative Splicing 2.2. Genetic Structure and Alternative Splicing Analysis The result of remapping the transcriptome reads indicated that all three sod genes possessed introns, and several intron regions were alternatively spliced (seethat Supplementary Figure M-6 to Figure The result of remapping the transcriptome reads indicated all three sod genes possessed introns, and several intron regions were alternatively spliced (see Supplementary Figure M-6 to M-8). The results are shown in Figure 1, and the sequences are shown in supplementary sequences S1 Figure M-8). The results are shown in Figure 1, and the sequences are shown in supplementary to S3. All the verified nucleotide sequences from this study have been submitted to GenBank under sequences S1 to S3. All the verified nucleotide sequences from this study have been submitted to the accession ID numbers KP747454, and KP747455. GenBank under the KP747453, accession ID numbers KP747453, KP747454, and KP747455.
Figure 1. Schematic representation of the V. volvacea sod gene structures.
Figure 1. Schematic representation of the V. volvacea sod gene structures.
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The Vv_Cu-Znsod1 Int. J. Mol. Sci. 2016, 17, 34 possessed eight introns, I1-I8, with lengths of 52, 76, 80, 125, 57, 58, 3 of54, 13 and 55 bp. Among them, intron retention existed for I1, I4, I7, and I8; I2 contained an alternative 3’ splice The Vv_Cu-Znsod1 possessed eight introns, I1-I8, with lengths ofThe 52, 76, 80, 125, 57,contained 58, 54, andthree site, and I3 showed either intron retention or alternative 3’ splicing. Vv_Mnsod1 55 bp. Among them, intron retention existed for I1, I4, I7, and I8; I2 contained an alternative 3’ splice introns (I1-I3) of 154, 57 and 56 bp, among which I1 and I2 had alternative splicing modes for intron site, and I3 showed either intron retention or alternative 3’ splicing. The Vv_Mnsod1 contained three retention, and I1 also had the 5’ alternative splice site. The Vv_Mnsod2 contained seven introns. The introns (I1-I3) of 154, 57 and 56 bp, among which I1 and I2 had alternative splicing modes for intron sizes retention, of I1–I7 were 72,had 126, 88, 61, and 87, site. among I1, I3, I4, I5, and I7 introns. had alternative and I147, also the119, 5’ alternative splice The which Vv_Mnsod2 contained seven The splicing sites for intron retention. sizes of I1–I7 were 47, 72, 126, 119, 88, 61, and 87, among which I1, I3, I4, I5, and I7 had alternative splicing sites for intron retention.
2.3. Alignment and Phylogenetic Analysis of Amino Acid Sequences 2.3. Alignment and Phylogenetic Analysis of Amino Acid Sequences As shown in Figure 2, 35 microorganism SOD sequences can be gathered into four branches of As MnSOD, shown inFeSOD, Figure 2, 35 NiSOD microorganism SOD sequences can be phylogenetic gathered into tree. four Among branchesthem, Cu-ZnSOD, and by the Neighbor-joining (NJ) of Cu-ZnSOD, MnSOD, FeSOD, and NiSOD by the Neighbor-joining (NJ) phylogenetic tree.were Volvariella volvacea_MnSOD1, Volvariella volvacea_MnSOD2, and Ganoderma microsporum_Q92429 Among them, Volvariella volvacea_MnSOD1, Volvariella volvacea_MnSOD2, and Ganoderma on the same branch, and the branch support degree was up to 86%. The Volvariella volvacea_Cu-ZnSOD1 microsporum_Q92429 were on the same branch, and the branch support degree was up to 86%. The and the Cu-ZnSODs of other fungi were on the same branch, with 85% of the support degree. The Volvariella volvacea_Cu-ZnSOD1 and the Cu-ZnSODs of other fungi were on the same branch, with tree revealed that, although attached to the Cu-ZnSOD branch, the Aspergillus japonicus_Q12548 was 85% of the support degree. The tree revealed that, although attached to the Cu-ZnSOD branch, the markedly different from the Cu-ZnSOD sequences of other fungi. Aspergillus japonicus_Q12548 was markedly different from the Cu-ZnSOD sequences of other fungi.
Figure 2. Neighbor-joining phylogenetic tree of SOD amino acid sequences across multiple species.
Figure 2. Neighbor-joining phylogenetic tree of SOD amino acid sequences across multiple species. Using the MUSCLE method to do the sequence alignment, the tree build was performed using the program Using the MUSCLE method to do the sequence alignment, the tree build was performed using MEGA version 5.1 and the “Partial Deletion” option to exclude positions covering less than 70% of the site. the program MEGA version 5.1 and the “Partial Deletion” option to exclude positions covering All of the sequences were downloaded from the UniProt database (http://www.uniprot.org/) except for less than 70% of the site. sequences were distance. downloaded from the thebranches UniProt database three V. volvacea SODs. (TheAll scaleof barthe indicates evolutionary Values above indicate (http://www.uniprot.org/) except for three V. volvacea SODs. (The scale bar indicates evolutionary the degree of bootstrap support from a 1000 replicate analysis. One Cu-ZnSOD and two MnSODs of V. distance. Values above by theblack branches the respectively.) degree of bootstrap support from a 1000 replicate volvacea were labeled circularindicate and triangle, analysis. One Cu-ZnSOD and two MnSODs of V. volvacea were labeled by black circular and Based on the findings of several studies [23,24], the Cu-ZnSOD and the MnSOD amino acid triangle, respectively.) sequences of Aspergillus fumigatus and Candida albicans were downloaded from the NCBI database, and the multiple sequence alignment of the three SOD amino acid sequences of the V. volvacea was Based on the findings of several studies [23,24], the Cu-ZnSOD and the MnSOD amino acid performed using the multiple sequence comparison by log-expectation (MUSCLE) method. As sequences and Candida albicans from NCBI database, shownof in Aspergillus Figure 3, thefumigatus results indicated that all three SODwere aminodownloaded acid sequences of V.the volvacea possess and the multiple sequence alignment of the three SOD amino acid sequences of the V. volvacea conserved amino acid residues involved in metal ion binding. The N terminus of the Vv_MnSOD2 was performed using multiple sequence comparison by log-expectation (MUSCLE) As in shown contains a 24 the amino acid residues mitochondrial targeting sequence (MTS), which is method. not detected
in Figure 3, the results indicated that all three SOD amino acid sequences of V. volvacea possess conserved amino acid residues involved in metal ion binding. The N terminus of the Vv_MnSOD2
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contains a 24Sci. amino in Int. J. Mol. 2016, 17,acid 34 residues mitochondrial targeting sequence (MTS), which is not detected 4 of 13 the Vv_MnSOD1. This observation indicates that the two MnSODs in V. volvacea play roles in the the Vv_MnSOD1. This observation indicates thatisthe two MnSODs in V.prediction volvacea play roles subcellular in the mitochondria and cytoplasm, respectively, which consistent with the of their mitochondria and cytoplasm, respectively, which is consistent with the prediction of their subcellular localization. According to its subcellular localization, the Vv_Cu-ZnSOD1 participates in the secretory localization. to its subcellular localization, the Vv_Cu-ZnSOD1 participates in 13the Int. J. Mol. Sci.According 2016, 17, 34 pathway. The prediction of the signal peptide also indicated that the Vv_Cu-ZnSOD1 has4 aof 17 amino secretory pathway. The prediction of the signal peptide also indicated that the Vv_Cu-ZnSOD1 has acid residues signal peptide sequenceindicates belonging to atwo secretory protein, which indicated Vv_MnSOD1. This observation thatbelonging the MnSODs in V. volvacea play rolesindicated in thethat the a 17the amino acid residues signal peptide sequence to a secretory protein, which Vv_Cu-Znsod1 gene encoding an extracellular Cu-ZnSOD protein (supplementary Figure S2).S2). mitochondria and cytoplasm, respectively, which is consistent with the prediction of their subcellular that the Vv_Cu-Znsod1 gene encoding an extracellular Cu-ZnSOD protein (supplementary Figure localization. According to its subcellular localization, the Vv_Cu-ZnSOD1 participates in the secretory pathway. The prediction of the signal peptide also indicated that the Vv_Cu-ZnSOD1 has a 17 amino acid residues signal peptide sequence belonging to a secretory protein, which indicated that the Vv_Cu-Znsod1 gene encoding an extracellular Cu-ZnSOD protein (supplementary Figure S2).
Figure 3. Multiple alignment of SOD amino acid sequences from V. volvacea (Volvo), A. fumigatus Figure 3. Multiple alignment of SOD amino acid sequences from V. volvacea (Volvo), A. fumigatus (Aspfu) and C. albicans (Canal). The sequences were downloaded from the NCBI database (Aspfu) and C. albicans (Canal). The sequences were downloaded from the NCBI database (http://www.ncbi.nlm.nih.gov/protein/) except for the V. volvacea SODs. The name (ID number) of (http://www.ncbi.nlm.nih.gov/protein/) except for the V. volvacea SODs. The name (ID number) of Cu-ZnSOD MnSOD sequences were marked the green andV.yellow respectively. The Figure 3. and Multiple alignment of SOD amino acidby sequences from volvaceaboxes, (Volvo), A. fumigatus Cu-ZnSOD andand MnSOD sequences were marked bywere the green and yellow boxes, respectively. The (Aspfu) C. albicans (Canal). The is sequences downloaded from the mitochondrial targeting sequence (MTS) boxed in purple; the metal binding sitesNCBI (H97, database H99, H114 mitochondrial targeting sequence (MTS) is boxed in purple; the metal binding sites (H97, H99, H114 volvacea SODs. name (ID number) of D134 for for the Zn2+V. ), C108, and C147The involved in formation of a and(http://www.ncbi.nlm.nih.gov/protein/) H171 for Cu2+; H114, H122, H131, andexcept 2+ ; H114, H122, H131, and D134 for Zn2+ ), C108, and C147 involved in formation of a and H171 for Cu Cu-ZnSOD sequences by glycosylation the green and site, yellow respectively. disulfide bondand andMnSOD N137, which actswere as amarked potential areboxes, highlighted with The green mitochondrial targeting sequence (MTS) is boxed in purple; the metal binding H99,green H114 [11,12]. disulfide bond N137, which acts as a potential site, are highlighted are highlighted in pink [13].sites (H97,with [11,12]. Theand potential metal-binding sites for Mn2+glycosylation 2+; H114, H122, H131, 2+ 2+), C108, and C147 involved in formation of a and D134 for Zn and H171 for Cu The potential metal-binding sites for Mn are highlighted in pink [13]. disulfideExpression bond and N137, as aShort-Term potential glycosylation 2.4. Differential of Sodwhich Genesacts under Cold Stresssite, are highlighted with green [11,12]. The potential metal-binding sites for Mn2+ are highlighted in pink [13].
2.4. Differential of Sod Genes underofShort-Term Stress To testExpression the transcriptional responses three Vv-sodCold genes to cold stress, the V. volvacea colonies 2.4. Differential Expression of Sodwere Genesswitched under Short-Term Stress temperature of 34 to 4 °C. After submerged in phosphate buffer from anCold incubation To test the transcriptional responses of three Vv-sod genes to cold stress, the V. volvacea colonies 1 h of cold stress, transcript levels of Vv_Cu-Znsod1, Vv_Mnsod1, and Vv_Mnsod2 in the cold-treated To test the transcriptional responses of three Vv-sod to cold stress, the V. volvacea submerged in phosphate buffer were switched from angenes incubation temperature of 34colonies to 4 ˝ C. After mycelia were increased by 5.1-fold, 4.6-fold, and 2.6-fold, respectively (Figure 4). submerged in phosphate buffer were switched from an incubation temperature of 34 to 4 °C. After
1 h of cold stress, transcript levels of Vv_Cu-Znsod1, Vv_Mnsod1, and Vv_Mnsod2 in the cold-treated 1 h of cold stress, transcript levels of Vv_Cu-Znsod1, Vv_Mnsod1, and Vv_Mnsod2 in the cold-treated myceliamycelia were increased by 5.1-fold, 4.6-fold, respectively (Figure were increased by 5.1-fold, 4.6-fold,and and2.6-fold, 2.6-fold, respectively (Figure 4). 4).
Figure 4. Differential expression of sod genes in V. volvacea H1521 stressed at low temperature for different times. Control (−), hyphae were incubated in phosphate buffered solution (PBS) buffer Figure 4. Differential expression of sod genes in V. volvacea H1521 stressed at low temperature for Figure 4. Differential expression in V. volvacea H1521 stressed at incubated low temperature (0.02 mol/L, pH 8, 34 °C) for 1.5of h sod at 34genes °C. Cold stress treated (+), hyphae were in PBS for different times. Control (−), hyphae were incubated in phosphate buffered solution (PBS) buffer buffer (0.02 mol/L, pH 8, 34 °C) for 0.5 h at 34 °C and then in PBS buffer (0.02 mol/L, pH 8, 4 °C) atbuffer different(0.02 times. hyphae were incubated phosphate buffered solution in(PBS) mol/L,Control pH 8, 34(´), °C) for 1.5 h at 34 °C. Cold stressintreated (+), hyphae were incubated PBS ˝ ˝ 4 °C for 1 h. Data are the averages of four independent experiments. The error bars show the standard (0.02 mol/L, 34 C) 1.5°C)h for at 34 Cold stress (+), hyphae werepHincubated buffer pH (0.028,mol/L, pHfor 8, 34 0.5 hC. at 34 °C and thentreated in PBS buffer (0.02 mol/L, 8, 4 °C) at in PBS ˝ C)analysis ˝ C andexperiments. error offor the mean. Statistical performed usingin least significant difference (LSD) 4 °C 1 h. Data of four The error bars show the standard buffer (0.02 mol/L, pHare 8,the 34 averages for 0.5 hwas atindependent 34 then PBS buffer (0.02 mol/L, pH 8,test, 4 ˝ C) at ** p < 0.01. ˝ of theare mean. performed experiments. using least significant difference (LSD)the test, 4 C forerror 1 h. Data the Statistical averagesanalysis of four was independent The error bars show standard p < 0.01. error of**the mean. Statistical analysis was performed using least significant difference (LSD) test, ** p < 0.01.
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2.5. Expression Int. J. Mol. Sci.Patterns 2016, 17, 34of Sod Genes during the Fruiting-body Development Stages of V. volvacea 5 of 13 All three SOD-encoding different levels 2.5. Expression Patterns of Sodgenes Genes showed during thesignificantly Fruiting-body Development Stagesof of expression V. volvacea in different growth stages of the fruiting body of V. volvacea. The statistical analysis indicated that the expression All three SOD-encoding genes showed significantly different levels of expression in different patterns of Vv_Cu-Znsod1 and Vv_Mnsod2 show similar expression patterns in different tissues of growth stages of the fruiting body of V. volvacea. The statistical analysis indicated that the expression V. volvacea. The correlation coefficient, 0.949, indicates a very good correlation [25]. Both genes show patterns of Vv_Cu-Znsod1 and Vv_Mnsod2 show similar expression patterns in different tissues of V. the highest of expression in the pileus of the egg stage andcorrelation are thought be correlated volvacea.level The correlation coefficient, 0.949, indicates a very good [25].to Both genes showwith the the morphogenesis pileus atinthis the Vv_Mnsod1 showed be less tissue-specific highest levelofofthe expression the stage. pileus However, of the egg stage and are thought to betocorrelated with the sincemorphogenesis the highest expression stage fruiting body) was only than the lowest of the pileustissue at this(button stage. However, the Vv_Mnsod1 showed to 5.6-fold be less tissue-specific since the highest tissue (button stage Although, fruiting body) only 5.6-fold the pileus lowest was expression tissue (theexpression stipe of elongation stage). thewas expression levelthan in the expression tissue (the stipe of elongation stage). Although, the expression level in the pileus wasgenes higher than in the stipe, the expression pattern of Vv_Mnsod1 was different from the other two sod higher than in the stipe, the expression pattern of Vv_Mnsod1 was different from the other two sod (correlation coefficients of 0.385 and 0.421) (Figure 5). Furthermore, the expression of Vv_Cu-Znsod1 genes (correlation coefficients of 0.385 and 0.421) (Figure 5). Furthermore, the expression of Vv_Cuand Vv_Mnsod2 in the lamellae showed good correlation with each other (correlation coefficient of Znsod1 and Vv_Mnsod2 in the lamellae showed good correlation with each other (correlation 1), and the level of expression in the egg stage was much higher than in the elongation stage or the coefficient of 1), and the level of expression in the egg stage was much higher than in the elongation maturation However, the However, expression Vv_Mnsod1 in the lamellae egg stage stage orstage. the maturation stage. theofexpression of Vv_Mnsod1 in the of lamellae of eggshowed stage no significant differences with elongation stage and maturation stage. (Figure showed no significant differences with elongation stage and maturation stage.6). (Figure 6).
Figure 5. Expression patterns of sod genes in different tissues during different developmental stages
Figure 5. Expression patterns of sod genes in different tissues during different developmental stages of V. volvacea H1521. (A) the different fruiting body growth stages of V. volvacea; (B) the differential of V. expression volvacea H1521. the different body growth stagespattern of V. volvacea; theand differential pattern(A) of Cu-Znsod1 gene;fruiting (C) the differential expression of Mnsod1(B) gene; (D) expression patternexpression of Cu-Znsod1 gene; (C) the differential expression pattern of EG Mnsod1 gene; and the differential pattern of Mnsod2 gene. PR = primordia; BU = button stage; = egg stage; (D) the of Mnsod2 gene. PRexpression = primordia; button stage; EGto= egg ELdifferential = elongationexpression stage; MA =pattern maturation stage. The gene levelsBU are=presented relative the MA-pileus. TheMA means of three independent are shown. The bars show stage;that EL in = elongation stage; = maturation stage. Theexperiments gene expression levels areerror presented relative theinstandard error of the mean, andofthe different letters over the columnsare within a graph to that the MA-pileus. The means three independent experiments shown. Thedenote error bars differences < 0.05, LSDand test,the n = different 3). showsignificant the standard error of(pthe mean, letters over the columns within a graph denote significant differences (p < 0.05, LSD test, n = 3).
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Figure 6. different developmental stages of V. Figure 6. Expression Expressionpatterns patternsofofsod sodgenes genesininlamellae lamellaeduring during different developmental stages of volvacea thethedifferential differential V. volvaceaH1521. H1521.(A)(A) differentialexpression expressionpattern patternofof Cu-Znsod1 Cu-Znsod1 gene; gene; (B): (B): the the differential expression pattern expression pattern of of Mnsod1 Mnsod1 gene; gene; and and (C) (C) the the differential differential expression expression pattern pattern of of Mnsod2 Mnsod2 gene. gene. EG = egg stage; EL = elongation stage; MA = maturation stage. The gene expression levels are EG = egg stage; EL = elongation stage; MA = maturation stage. The gene expression levels are presented presented relative to the lamellae of MA stage. The means of four independent experiments are relative to the lamellae of MA stage. The means of four independent experiments are shown. The error shown. The error bars show standard error of the mean. was Statistical analysis was performed bars show the standard error the of the mean. Statistical analysis performed using LSD test, ** p
