Asexual reproductive organ-specific expression of the glyceraldehyde-3-phosphate dehydrogenase 2 gene of Pilobolus crystallinus

Pilobolus crystallinus has three putative glyceraldehyde-3-phosphate dehydrogenase (gapdh) genes (pcgapdh1, pcgapdh2 and pcgapdh3). The results of this study demonstrate that expression of pcgapdh2 was increased by irradiation and that this increased expression was correlated with the formation of asexual reproductive organs (trophocysts). Interestingly, expression of pcgapdh2 was restricted to trophocysts. The formation of trophocysts was likely promoted by light, and the expression of pcgapdh2 was increased as a result of trophocyst formation. This is the first report that shows the regulation of a gapdh gene in an organ-specific manner in fungi.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Mucor—a source of cocoa butter and gamma-linolenic acid

Lipid analyses were performed on 28 strains of various species of the genus Mucor. In shake flasks with glucose as carbon source, the gamma-linolenic acid (GLA) content in the neutral lipid (NL) fraction of some Mucor species was up to 38 mg GLA/g dry biomass. Some Mucor species produced more than 20% (w/w) stearic acid (18:0) and more than 60% of their NL content as symmetrical triacylglycerols (SUS-TAGs) which corresponded to those of cocoa butter. Three Mucor species were evaluated in terms of the production of SUS-TAGs and GLA in pH-stat, fed-batch cultures in an air-lift fermenter with acetic acid as titrant and carbon source. Mucor circinelloides f. circinelloides CBS 108.16 accumulated 27% 18:0 in the NL fraction, which constituted approximately 40% of the dry biomass. In this case, the NL fraction contained more than 70% (w/w) SUS-TAGs.M.P. Roux, J.L.F. Kock, A. Botha, J.C. du Preez and P.J. Botes are with the Department of Microbiology and Biochemistry, University of the Orange Free State, P.O. Box 339, Bloemfontein 9300, South Africa; G.V. Wells is with Sasol Waxes, P.O. Box 1, Sasolburg, South Africa.     

Synergistic interaction of glyceraldehydes‐3‐phosphate dehydrogenase and ArsJ,a novel organoarsenical efflux permease,confers arsenate resistance

Microbial biotransformations are major contributors to the arsenic biogeocycle. In parallel with transformations of inorganic arsenic, organoarsenicals pathways have recently been recognized as important components of global cycling of arsenic. The well‐characterized pathway of resistance to arsenate is reduction coupled to arsenite efflux. Here, we describe a new pathway of arsenate resistance involving biosynthesis and extrusion of an unusual pentavalent organoarsenical. A number of arsenic resistance (ars) operons have two genes of unknown function that are linked in these operons. One, gapdh, encodes the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase. The other, arsJ, encodes a major facilitator superfamily (MFS) protein. The two genes were cloned from the chromosome of Pseudomonas aeruginosa. When expressed together, but not alone, in Escherichia coli, gapdh and arsJ specifically conferred resistance to arsenate and decreased accumulation of As(V). Everted membrane vesicles from cells expressing arsJ accumulated As(V) in the presence of purified GAPDH, D‐glceraldehylde 3‐phosphate (G3P) and NAD⁺. GAPDH forms the unstable organoarsenical 1‐arseno‐3‐phosphoglycerate (1As3PGA). We propose that ArsJ is an efflux permease that extrudes 1As3PGA from cells, where it rapidly dissociates into As(V) and 3‐phosphoglycerate (3PGA), creating a novel pathway of arsenate resistance.     

Electrophoretic karyotypes of some related Mucor species

Contour clamped homogeneous electric field (CHEF) gel electrophoresis was used to obtain electrophoretic karyotypes from nine Mucorstrains representing five different species (M. bainieri, M. circinelloides, M. mucedo, M. plumbeus and M. racemosus). The chromosomal banding patterns revealed high variability among the isolates. The sizes of the DNA in the Mucor chromosomes were estimated to be between 2.5 and 8.7 Mb. The total genome sizes were calculated to be between 30.0 and 44.7 Mb. The applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation is considered.     

产甘油假丝酵母与酿酒酵母胞浆3-磷酸甘油脱氢酶基因的功能比较

胞浆3-磷酸甘油脱氢酶(GPD)是酿酒酵母细胞甘油合成过程中的关键限速酶．尽管高产甘油菌株产甘油假丝酵母基因组中编码该酶的基因CgGPD已经被克隆出来,但是具体的功能,特别是与酿酒酵母GPD1和GPD2基因的功能比较值得进一步研究．以酿酒酵母渗透压敏感型的gpd1/gpd2和gpd1突变株为宿主,分别导入CgGPD、GPD1和GPD2基因,比较分析了CgGPD、GPD1和GPD2基因在高渗透压胁迫条件下和厌氧环境中的表达调控,及其对细胞甘油合成能力的影响．研究发现,GPD1基因受到渗透压诱导表达,GPD2基因在细胞厌氧条件下起着氧化还原平衡调节作用,而CgGPD基因不仅能够在渗透压胁迫条件下通过过量快速合成甘油调节渗透压平衡,而且能够在厌氧培养环境中互补GPD2基因的缺失,使gpd1/gpd2缺失突变株能够正常生长,同时提高了突变株的甘油合成能力．结果表明,CgGPD基因在gpd1/gpd2缺失突变株中既具有GPD1基因的功能,又能发挥GPD2基因的功能．     

水玉霉(Pilobolus)属的种的重新划分

我们在过去的工作中承认水玉霉(Pilobolus)属的9个种(郑、胡,见戴,1979)。近年来我们重新分离得到了这些分类群并对它们进行了再研究。研究结果表明,尽管它们是彼此可以互相区分的分类群,但是,包括我们过去的概念在内,目前被普遍接受的用于这个属的分类的种概念太小.为了与整个毛霉目的其它属的分类系统相一致,我们把这9个分类群重新划分为由9个变种组成的5个种:晶澈水玉霉原变种[Pilobolus crystallinu (Wigg.) Tode var. crystallinus],晶澈水玉霉透孢变种新组合[P. crystallinus var.hyalosporus (Boedijn) Hu &Zheng, comb. nov.],晶澈水玉霉克莱因变种新组合[P. crystallinus var.kleinii (van Tieghem) Zheng &G.-q. Chen, comb. nov.],豆状水玉霉原变种(P. lentiger Corda var. lentiger),豆状水玉霉小型变种新组合[P. lentiger var. minutus (Speg.) Zheng &G.-q. Chen, comb. nov.],长型水玉霉(P. longipes van Tieghem),厚壁水玉霉(P. Oedipus Mont.),露水玉霉原变种[P. roridus (Bolt.) Pers. var. roridus],露水玉霉突囊变种新组合[P. roridus var. umbonatus (Butler) Hu &Zheng, comb. nov.]。水玉霉属先后报道过的种或种下分类群名称共计50个左右,其中一些异名往往被不同的作者归到不同的正名下面。为了解决它们的正确归属问题,我们对全部原始描述作了细致的文献考证然后决定其位置。对那些找不到原始描述或从原始描述中得不出结论的则作为可疑名称处理。可疑名称共计12个:Mucor obliquus Scop., M. urceolatus Dicks.;Pilobolus urceolatus Purt., P. pestis-bovinae Hallier(=P. hallierii Rivolta), P. nanus van Tieghem, P. intermedius, (Coem.) P. A. Karsten(=P. Oedipus Mont. var,intermedius Coem.), P. pullus Massee, P.proliferens McVickar, P. ramosus McVickar, P. simplex McVickar, P. lentiger forma leinii Reyn. &Laysa, P. lentiger forma minutus Reyn. &Laysa.     

Overexpression of Wheat <i>TaELF3-1BL</i> Delays Flowering in Arabidopsis

EARLY FLOWERING 3 (ELF3), a light zeitnehmer (time-taker) gene, regulates circadian rhythm and photoperiodic flowering in Arabidopsis, rice, and barley. The three orthologs of ELF3 (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) have been identified in wheat too, and one gene, TaELF3-1DL, has been associated with heading date. However, the basic characteristics of these three genes and the roles of the other two genes, TaELF3-1BL and, TaELF3-1AL, remain unknown. Therefore, the present study obtained the coding sequences of the three orthologs (TaELF3-1AL, TaELF3-1BL, and TaELF3-1DL) of ELF3 from bread wheat and characterized them and investigated the role of TaELF3-1BL in Arabidopsis. Protein sequence comparison revealed similarities among the three TaELF3 genes of wheat; however, they were different from the Arabidopsis ELF3. Real-time quantitative PCR revealed TaELF3 expression in all wheat tissues tested, with the highest expression in young spikes; the three genes showed rhythmic expression patterns also. Furthermore, the overexpression of the TaELF3-1BL gene in Arabidopsis delayed flowering, indicating their importance in flowering. Subsequent overexpression of TaELF3-1BL in the Arabidopsis ELF3 nonfunctional mutant (elf3 mutant) eliminated its early flowering phenotype, and slightly delayed flowering. The wild-type Arabidopsis overexpressing TaELF3-1BL demonstrated reduced expression levels of flowering-related genes, such as CONSTANS (AtCO), FLOWERING LOCUS T (AtFT), and GIGANTEA (AtGI). Thus, the study characterized the three TaELF3 genes and associated TaELF3-1BL with flowering in Arabidopsis, suggesting a role in regulating flowering in wheat too. These findings provide a basis for further research on TaELF3 functions in wheat.     

Identification,characterization and interaction of <Emphasis Type="Italic">HAP</Emphasis> family genes in rice

A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5 genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB288027 to AB288048 and BR000373 to BR000375.     

Aerobic and anaerobic ethanol production by<Emphasis Type="Italic"> Mucor circinelloides</Emphasis> during submerged growth

The dimorphic organism Mucor circinelloides is currently being investigated as a potential host for heterologous protein production. The production of ethanol on pentose and hexose sugars was studied in submerged batch cultivations to further the general knowledge of Mucor physiology, with a view to the minimisation or elimination of the by-product ethanol for future process design. Large amounts of ethanol were produced during aerobic growth on glucose under non-oxygen limiting conditions, which is indicative of M. circinelloides being a Crabtree-positive organism. Ethanol production on galactose or xylose was less significant. The response of the organism to increased ethanol concentrations, both as the sole carbon source and in the presence of a sugar, was investigated in terms of biomass formation and morphology.     

The RNAi machinery controls distinct responses to environmental signals in the basal fungus Mucor circinelloides

Background

RNA interference (RNAi) is a conserved mechanism of genome defence that can also have a role in the regulation of endogenous functions through endogenous small RNAs (esRNAs). In fungi, knowledge of the functions regulated by esRNAs has been hampered by lack of clear phenotypes in most mutants affected in the RNAi machinery. Mutants of Mucor circinelloides affected in RNAi genes show defects in physiological and developmental processes, thus making Mucor an outstanding fungal model for studying endogenous functions regulated by RNAi. Some classes of Mucor esRNAs map to exons (ex-siRNAs) and regulate expression of the genes from which they derive. To have a broad picture of genes regulated by the silencing machinery during vegetative growth, we have sequenced and compared the mRNA profiles of mutants in the main RNAi genes by using RNA-seq. In addition, we have achieved a more complete phenotypic characterization of silencing mutants.

Results

Deletion of any main RNAi gene provoked a deep impact in mRNA accumulation at exponential and stationary growth. Genes showing increased mRNA levels, as expected for direct ex-siRNAs targets, but also genes with decreased expression were detected, suggesting that, most probably, the initial ex-siRNA targets regulate the expression of other genes, which can be up- or down-regulated. Expression of 50% of the genes was dependent on more than one RNAi gene in agreement with the existence of several classes of ex-siRNAs produced by different combinations of RNAi proteins. These combinations of proteins have also been involved in the regulation of different cellular processes. Besides genes regulated by the canonical RNAi pathway, this analysis identified processes, such as growth at low pH and sexual interaction that are regulated by a dicer-independent non-canonical RNAi pathway.

Conclusion

This work shows that the RNAi pathways play a relevant role in the regulation of a significant number of endogenous genes in M. circinelloides during exponential and stationary growth phases and opens up an important avenue for in-depth study of genes involved in the regulation of physiological and developmental processes in this fungal model.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1443-2) contains supplementary material, which is available to authorized users.     

Molecular characteristics and expression profiles of glycerol-3-phosphate dehydrogenase 1 (GPD1) gene in pig

The cytosolic activity of glycerol-3-phosphate dehydrogenase 1 (GPD1, EC 1.1.1.8) plays an important role in the synthesis of triacylglycerol and in the transport of reducing equivalents from the cytosol to mitochondria. Here we report the full-length genomic sequence of porcine GPD1 gene including promoter region. Porcine GPD1 gene contains eight exons and seven introns. Using the ImpRH, the GPD1 gene was mapped on chromosome 5. Sub-cellular localization of the pig GPD1 was localized in cytoplasm by GFP reporter gene. The full-length CDS of porcine GPD1 gene comprises 1050 nucleotides and it encodes 349 amino acids. Using the CDS sequences of 17 species, we built the phylogeny tree of GPD1 gene. We investigated the expression level of the gene in 13 different tissues and time course from birth to postnatal day 28 in longissinus doris muscle (LD) and in cerebrum. The result shows that porcine GPD1 gene is expressed in almost all tissues we tested but its levels of expression varies widely over 2 orders of magnitude. LD and the cerebrum have similar expression pattern that is at a low level at birth and increasing with aging to the highest level at postnatal day 8 in LD and postnatal day 14 in cerebrum. But weaning decreased the expression level of the GPD1 gene. This may partially explains the effects of weaning on energy metabolism.     

Improvement in Oil Production by Increasing Malonyl-CoA and Glycerol-3-Phosphate Pools in Scenedesmus quadricauda

In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes (ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0546-4) contains supplementary material, which is available to authorized users.