Cloning and characterisation of the glyceraldehyde 3-phosphate dehydrogenase gene of Candida bombicola and use of its promoter

The glyceraldehyde-3-phosphate dehydrogenase gene (GPD) of the sophorolipid producing yeast Candida bombicola was isolated using degenerated PCR and genome walking. The obtained 3,740 bp contain the 1,008 bases of the coding sequence and 1,613 and 783 bp of the upstream and downstream regions, respectively. The corresponding protein shows high homology to the other known GPD genes and is 74% identical to the gyceraldehyde-3-phosphate dehydrogenase of Yarrowia lipolytica. The particular interest in the C. bombicola GPD gene sequence originates from the potential use of its promoter for high and constitutive expression of homologous and heterologous genes. Southern blot analysis did not give any indication for the presence of multiple GPD genes and it can therefore be expected that the promoter can be used for efficient and high expression. This hypothesis was further confirmed by the biased codon usage in the GPD gene. GDP promoter fragments of different lengths were used to construct hygromycin resistance cassettes. The constructs were used for the transformation of C. bombicola and all of them, even the ones with only 190 bp of the GPD promoter, were able to render the cells resistant to hygromycin. The efficacy of a short GPD promoter can be a convenient characteristic for the construction of compact expression cassettes or vectors for C. bombicola. The GenBank accession number of the sequence described in this article is EU315245.     

PEG-mediated Transformation of Lentinus edodes

Expression vector p301-bG1 contains a gus gene and a bialaphos resistance gene both driven by glyceraldehydes-3-phosphate dehydrogenase (GPD) gene promoter isolated from Lentinus edodes (Berk.) Sing. Using p301-bG1, PEG-mediated transformation of protoplast of L. edodes was studied. Mixed with PEG-purified plasmid DNA, the protoplasts of L. edodes were treated with PEG solution and cultured on CYM regeneration plate containing 40 μg/mL bialaphos. Bialaphos-resistant and GUS-positive transformants were obtained using this transformation system. Although the transformation efficiency was relatively low, the protocols release large expenses on expensive instrument and restriction enzymes, providing a simple and economical method for mushroom breeding at the molecular level.     

Construction of a homologous selectable marker gene for Lentinula edodes transformation

We cloned a gene for the iron sulfur protein (Ip) subunit from an edible mushroom, Lentinula edodes, and introduced a point mutation that confers carboxin resistance into it. The mutant gene successfully transformed L. edodes with high efficiency (9 transformants/2.5 microg vector DNA). Restriction enzyme-mediated integration (REMI) increased the transformation efficiency by about two-fold.     

A transformation system for an ectomycorrhizal basidiomycete, Lyophyllum shimeji

A transformation vector, pLS-hph, was constructed with the promoter and terminator of the glyceraidehyde-3-phosphate dehydrogenase (GPD) gene derived from an ectomycorrhizal basidiomycete, Lyophyllum shimeji, and with the hygromycin B (HmB) phosphotransferase (hph) gene from Escherichia coli. This vector was introduced into protoplasts of L. shimeji and 3.4 transformants per microg plasmid DNA were obtained. In most of the transformants, multiple copies of the vector were integrated into the genomic DNA. The results indicate that pLS-hph is a useful vector for L. shimeji.     

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.     

Transformation of taxol-producing endophytic fungi by restriction enzyme-mediated integration (REMI)

The REMI method was used to introduce the plasmid pV2 harboring the hygromycin B phosphotransferase (hph) gene controlled by the Aspergillus nidulans trpC promoter and the trpC terminator into a taxol-producing endophytic fungus BT2. REMI transformation yielded stable transformants capable of continuing to grow on PDA medium containing 125 mug mL(-1) hygromycin B. The transformation efficiency was about 5-6 transformants mug(-1) plasmid DNA. The presence of hph gene in transformants was confirmed by PCR and Southern blot analyses. To the authors' knowledge, this is the first report on the transformation of taxol-producing endophytic fungi by the REMI technique. This study provides an effective approach for improving taxol production of endophytic fungi by the genetic engineering of taxol biosynthetic pathway genes in the future.     

A reliable transformation method and heterologous expression of beta-glucuronidase in Lentinula edodes

A simple and reliable mushroom transformation procedure based on electroporation of basidiospores or mycelial fragments was developed. This method eliminated the problem of protoplast preparation, the transformation efficiency were 30-150 transformants per mug DNA and the hygromycin resistant marker gene and gus were expressed in Lentinula edodes successfully. No false positive antibiotic-resistant cultures were detected by PCR amplification and the beta-glucuronidase (GUS) expression was maintained stable during mitotic cell division without selection pressure for more than 6 months. Southern analysis of transformants indicated the integration of gene might occur by non-homologous recombination. Using the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter with the first intron of gpd gene, the average GUS activity in L. edodes reached 144.6+/-3.9 U mg(-1) soluble protein, while only 30.1+/-0.7 U mg(-1) soluble protein was detected for those without the intron. The percentage of GUS in total soluble protein was 5.67 x 10(-4) (0.06%) for the transformant with the highest GUS activity. This rapid and convenient electroporation procedure offers a new approach for the genetic manipulation and tool to tag genes of important edible mushroom species.     

红曲霉不同转化方法比较

为了研究红曲霉聚酮体途径,考察和比较了4种不同的转化方法以建立有效的红曲霉遗传转化系统。以潮霉素作为抗性筛选标记,pBC-Hygro作为转化载体,用基于原生质体的传统转化和电击转化、基于萌发孢子的电击转化以及REMI技术转化红曲霉。发现基于萌发孢子的电击转化由于转化率极低而不适于红曲霉转化。基于原生质体的传统转化和电击转化尽管每微克DNA分别能获得135个转化子和125个转化子,但因转化子稳定性差也适合红曲霉转化的转化。应用REMI技术,转化率提高约20倍,每微克DNA 2500个转化子,70%～75%的转化子的稳定,非常适合于红曲霉的转化。      

Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis.

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA12-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

Deletions in the Gibberellin Biosynthesis Gene Cluster of Gibberella fujikuroi by Restriction Enzyme-Mediated Integration and Conventional Transformation-Mediated Mutagenesis

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA₁₂-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

过量表达大叶补血草LgNHX1基因对拟南芥耐盐性的影响

利用植物表达载体pCAMBIA1301和农杆菌GV3101将LgNHX1(全长1 656 bp)基因在拟南芥中过量表达.在含30 mg/L潮霉素的培养基上筛选获得LgNHX1的纯合转化子,并对其进行了分子鉴定和耐盐性分析.结果显示,经PCR和RT-PCR鉴定,野生型植株(对照)没有出现扩增条带,而转基因株系有相应的扩增条带,表明LgNHX1的确已经整合到拟南芥的基因组中,并已正常转录.在不同盐浓度处理下,转基因株系生长情况好于野生型对照;转基因植株地上部分和根的干重、鲜重相对高于野生型对照,但差异没有达到显著水平;当盐浓度达到150-200 mmol/L时,两个特基因株系的Na+含量显著高于野生型,K+含量极显著高于野生型.以上结果表明,过量表达LgNHX1基因可能增强了拟南芥将Na+区隔化至液泡的能力,提高了转基因拟南芥的耐盐能力.     

Mutagenesis via insertional- or restriction enzyme-mediated-integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic fungi.

Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Improved degradation of organophosphate dichlorvos by Trichoderma atroviride transformants generated by restriction enzyme-mediated integration (REMI)

A simple technique, REMI (restriction enzyme-mediated integration), was used to construct transformants of Trichoderma atroviride with improved capability of degrading organophosphate pesticide dichlorvos. Linearized DNA of plasmid pV2 bearing the hygromycin B phosphotransferase (hph) gene was inserted into chromosomes of wild strain T23 and transformation was confirmed by PCR and Southern blot analysis, respectively. Of 247 transformants, 76% showed improved dichlorvos degradation ability as compared to the parent strain T23 based on the least significant difference (LSD) test at p=0.01. Among them, 8 transformants exhibited 30% higher in degradation rate than the parent isolate. The highest dichlorvos degradation rate of the transformants was up to 96%. This study provided an effective approach for improving organophosphate pesticide-degrading capability of T. atroviride.     

Transformation frequencies are enhanced and vector DNA is targeted during retransformation of Leptosphaeria maculans, a fungal plant pathogen.

Summary Leptosphaeria maculans, a fungal pathogen of Brassica spp., was successfully transformed with the vector pAN8-1, encoding phleomycin resistance. Protoplasts of a vigorous Phleo^r transformant were then retransformed using the partially homologous vector, pAN7-1 which encodes hygromycin B resistance. Retransformation of this strain to hygromycin resistance occurred at frequencies that were consistently twofold higher than with the original recipient strain. Linearised pAN7-1 DNA transformed phleomycin-resistant protoplasts at higher frequencies still. All the transformants that were tested retained a phleomycin-resistant phenotype (20/20). Molecular analysis of five transformants generated with circular pAN7-1 DNA indicated that in four cases the pAN7-1 vector had integrated into pAN8-1 sequences. These results suggest that transformation frequencies in L. maculans are limited by the ability of vector DNA to integrate into the genome. Hence, construction of strains with target sites for integration may prove to be a generally useful method for improving transformation frequencies of poorly characterised filamentous fungi, particularly when using heterologous vectors. This would greatly facilitate the identification of genes by transfer of gene libraries and the standardisation of chromosomal location effects in studies of expression of nested promoter deletions.     

Structure of the cutinase gene and detection of promoter activity in the 5''-flanking region by fungal transformation.

The cutinase gene from Fusarium solani f. sp. pisi (Nectria hematococa) was cloned and sequenced. Sau3A fragments of genomic DNA from the fungus were cloned in a lambda Charon 35 vector. When restriction fragments generated from the inserts were screened with 5' and 3' probes from cutinase cDNA, a 5.5-kilobase SstI fragment hybridized with both probes, suggesting the presence of the entire cutinase gene. A 2,818-base pair segment was sequenced, revealing a 690-nucleotide open reading frame that was identical to that found in the cutinase cDNA with a single 51-base pair intron. Transformation vectors were constructed containing a promoterless gene for hygromycin resistance, which was translationally fused to flanking sequences of the cutinase gene. When protoplasts and mycelia were transformed with these vectors, hygromycin-resistant transformants were obtained. Successful transformation was assessed by Southern blot analysis by using radiolabeled probes for the hygromycin resistance gene and the putative promoter. The results of Southern blot analysis indicated that the plasmid had integrated into the Fusarium genome and that the antibiotic resistance was a manifestation of the promoter activity of the cutinase flanking sequences. Transformation of Colletotrichum capsici with the same construct confirmed the promoter activity of the flanking region and the integration of the foreign DNA. Transformation and deletion analysis showed that promoter activity resided within the 360 nucleotides immediately 5' to the cutinase initiation codon.