RESUMO
Overexpression of Dunaliella parva (D. parva) malic enzyme (ME) gene (DpME) significantly increased DpME expression and ME enzyme activity in transgenic D. parva. Nitrogen limitation had an inhibitory effect on protein content, and DpME overexpression could improve protein content. Nitrogen limitation increased carbohydrate content, and Dunaliella parva overexpressing malic enzyme gene under nitrogen limitation (DpME-N-) group showed the lowest starch content among all groups. Dunaliella parva overexpressing malic enzyme gene under nitrogen sufficient condition (DpME) and DpME-N- groups showed considerably high mRNA levels of DpME. ME activity was significantly enhanced by DpME overexpression, and nitrogen limitation caused a smaller increase. DpME overexpression and nitrogen limitation obviously enhanced lipid accumulation, and DpME overexpression had more obvious effect. Compared with control (wild type), lipid content (68.97%) obviously increased in DpME-N- group. This study indicated that the combination of DpME overexpression and nitrogen limitation was favorable to the production of microalgae biodiesel.
RESUMO
A homology-based PCR method was used to clone a cDNA encoding lanosterol synthase (LS) from Ganoderma lucidum (G. lucidum), which produces triterpenes. The cDNA of the LS (GenBank accession no. GQ169528) was found to contain an open reading frame (ORF) of 2,181 bp encoding a 726 amino acid polypeptide, whereas the LS genomic DNA sequence (GenBank accession no. GQ169529) consists of 2,924 bp. Functional complementation of G. lucidum LS (Gl-LS) in an erg7 yeast strain lacking LS activity demonstrated that the cloned cDNA encoded a functional LS. Analysis of the Gl-LS transcript profiles revealed a positive correlation between the pattern of LS gene expression and triterpenes content changes in G. lucidum during development. Up-regulation of expression of the Gl-LS gene by methyl jasmonate (MeJA) in the mycelia was also demonstrated by real-time RT-PCR. Up-regulation of the Gl-LS promoter activity by MeJA was also investigated.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Transferases Intramoleculares/genética , Reishi/enzimologia , Reishi/genética , Acetatos/farmacologia , Sequência de Aminoácidos , Clonagem Molecular , Ciclopentanos/farmacologia , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Dados de Sequência Molecular , Oxilipinas/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Reishi/crescimento & desenvolvimento , Reishi/metabolismo , Saccharomyces cerevisiae/genética , Triterpenos/metabolismoRESUMO
A farnesyl-diphosphate synthase gene, designated GlFPS, was isolated from a triterpene-producing basidiomycetous fungus, Ganoderma lucidum. The GlFPS cDNA was found to contain an open reading frame of 1,083 bp, encoding a protein of 360 amino acids with a calculated molecular mass of 41.27 kDa. The deduced amino acid sequence of the GlFPS cDNA exhibited a high homology with other fungal FPS genes, and contained four conserved domains. Phylogenetic analysis showed that GlFPS belonged to the basidiomycete FPS group. Competitive PCR revealed that GlFPS was constitutively expressed in the mycelium growth stage, whereas the transcripts of GlFPS accumulated to high levels rapidly during the process of mushroom primordia. Treatment of mycelia with exogenous methyl jasmonate also caused a large accumulation of GlFPS mRNA. Subsequently, promoter analysis indicated that the 5' upstream region of GlFPS possessed various potential regulatory elements associated with physiological and environmental factors. Functional complementation of GlFPS in an ERG20-disrupted yeast strain indicated that the cloned cDNA encoded a farnesyl-diphosphate synthase.
Assuntos
Expressão Gênica , Geraniltranstransferase/metabolismo , Reishi/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Geraniltranstransferase/genética , Geraniltranstransferase/isolamento & purificação , Dados de Sequência Molecular , Filogenia , Reishi/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
A gene encoding 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) was isolated from a triterpene-producing fungus, Ganoderma lucidum (Reishi or Lingzhi). This report provides the complete nucleotide sequence of the full-length cDNA encoding HMGR and its genomic DNA sequence. The cDNA of the HMGR (GenBank Accession no., EU263989) was found to contain an open reading frame (ORF) of 3,681 bp encoding a 1,226-amino-acid polypeptide, whereas the HMGR genomic DNA sequence (GenBank Accession no., EU263990) consisted of 4,262 bp and contained seven exons and six introns. The deduced amino acid sequence of G. lucidum HMGR showed significant homology to the known HMGRs from Ustilago maydis and Cryptococcus neoformans, and contained four conserved domains. Gene expression analysis showed that the expression level was relatively low in mycelia incubated for 10, 12, and 14 d, and reached the highest level in the primordia. Functional complementation of Gl-HMGR in a HMGR-deficient mutant yeast strain indicated that the cloned cDNA encoded a HMG-CoA reductase.