Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Biochemical characterization of chitinase 2 expressed during the autolytic phase of the inky cap, Coprinellus congregatus

Fungal cell walls consist of various glucans and chitin. The inky cap, Coprinellus congregatus, produces mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom is autolyzed rapidly to generate black liquid droplets in which no cell walls are detected by microscopy. Chitinase cDNA from the mature mushroom tissues of C. congregatus, which consisted of 1,622 nucleotides (chi2), was successfully cloned using the rapid amplification of cDNA ends polymerase chain reaction technique. The deduced 498 amino acid sequence of Chi2 had a conserved catalytic domain as in other fungal chitinase family 18 enzymes. The Chi2 enzyme was purified from the Pichia pastoris expression system, and its estimated molecular weight was 68 kDa. The optimum pH and temperature of Chi2 was pH 4.0 and 35°C, respectively when 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was used as the substrate. The K _m value and V _max for the substrate A, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside, was 0.175 mM and 0.16 OD min^?1unit^?1, respectively.     

Stress-induced expression of cucumber chitinase and <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis><Emphasis Type="Italic">β</Emphasis>-1,3-glucanase genes in transgenic potato plants

The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) cultivar ETA using Agrobacterium tumefaciens. Expression of both genes was driven by wound-inducible polyubiquitin promoter isolated from Slovak potato breeding line 116/86. Analyses showed inducible, peel-specific expression of both transgenes under stress conditions. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro and in vivo. Experiments with crude protein extracts isolated from transgenic microtubers showed growth inhibition of Rhizoctonia solani hyphae in the range from 7.3 to 14.2%. In contrast, experiments performed in growth chamber conditions revealed that the polyubiquitin promoter driven transgene expression did not ensure any obvious increase of transgenic potato resistance against Rhizoctonia solani.     

Antifungal chitinase against human pathogenic yeasts from Coprinellus congregatus

The inky cap, Coprinellus congregatus, produces mushrooms which become autolyzed rapidly to generate black liquid droplets, in which no cell wall is detected by microscopy. A chitinase (Chi2) which is synthesized during the autolytic phase of C. congregatus inhibits the growths of Candida albicans and Cryptococcus neoformans up to 10% at the concentration of 10 μg/ml, about 50% at concentration of 20 μg/ml, and up to 95% at the concentration of 70 μg/ml. Upon treatment these yeast cells are observed to be severely deformed, with the formation of large holes in the cell wall. The two yeast species show no growth inhibition at the concentration of 5 μg/ml, which means the minimum inhibitory concentrations for both yeast species are 10 μg/ml under these experimental conditions.     

Enhanced expression of chitinase during the autolysis of mushroom in <Emphasis Type="Italic">Coprinellus congregatus</Emphasis>

Fungal cell walls consist of various glucans and chitin. An inky cap, Coprinellus congregates, produced mushrooms at 25°C in a regime of 15 h light/9 h dark, and then the mushroom was autolyzed rapidly to generate black liquid droplets where no cell wall was detected by microscopy. A chitinase cDNA from the matured mushroom cells of C. congregates that consisted of 1,541 nucleotides was successfully cloned using the rapid amplification of cDNA ends (RACE)-PCR technique. Its deduced 441 amino acid sequence had the conserved catalytic domain as in other fungal chitinase family 18. Chitinase activity was higher at the matured mushroom stage than primordial and young mushroom stage. When the expression of the cloned chitinase was examined by real-time PCR using the chitinase-specific primers, it was increased more than twice to 20 times during the autolytic process of mushroom than young mushroom or primordial stages, respectively.     

Co-bombardment,integration and expression of rice chitinase and thaumatin-like protein genes in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> cv. Conlon)

Pathogenesis-related (PR) proteins associated with degradation of structural components of pathogenic filamentous fungi were overexpressed in the two-rowed malting barley (Hordeum vulgare L.) cultivar Conlon. Transgenes were introduced by co-bombardment with two plasmids, one carrying a rice (Oryza sativa L.) chitinase gene (chi11) and another carrying a rice thaumatin-like protein gene (tlp). Each gene was under the control of the maize ubiquitin (Ubi1) promoter. Fifty-eight primary transformants from three independent transformation events were regenerated. T₁ plants with high rice chi11 and tlp protein expression levels were advanced to identify T₂ homozygotes by herbicide spray and subjected to further molecular analyses. T₃ progeny from one event (E2) had stable integration and expression of the rice chi11 and tlp while those from the other events (E1 and E3) showed stable integration only of tlp. The successful production of these lines overexpressing the antifungal chi and tlp proteins provides materials to test the effects of these genes on a variety of fungal diseases that attack barley and to serve as potential additional sources of disease resistance.     

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant protein production by GAL10 promoter using a lipase, Candida antarctica lipase B (CalB), as a reporter. While the use of Δgal1 mutant strain required the addition of a certain amount of galactose to the medium, Δgal80 mutant strain did not require galactose. Furthermore, it was found that the recombinant CalB could be produced more efficiently (1.6-fold at 5 L-scale fermentation) in Δgal80 mutant strain than in the Δgal1 mutant. The Δgal80 mutant strain showed glucose repressible mode of expression of GAL10 promoter. Using Δgal80 mutant strain of S. cerevisiae, CalB was efficiently produced in a glucose-only fermentation at volumes up to 500 L.     

A Novel Culture Method for High Level Production of Heterologous Protein in Saccharomyces cerevisiae

A high level production system for heterologous protein by cold culture of yeast transformants at 15°C was developed. The yeast transformants, carrying a plasmid containing cDNA for Aspergillus oryzae α-amylase (Taka-amylase A) or human lysozyme synthetic DNA, were cultivated in a selective medium for 1 or 2 days until full growth at 30°C. The yeast cells were harvested by centrifugation from the culture fluid and then were transferred to YPD medium. These inoculated broths were incubated for 2 days at 15°C and then for another 2 days at 30°C. By the cold culture method described above, higher amounts of Taka-amylase A (28.6 mg/liter) and human lysozyme (6.1 mg/liter) were produced by the yeast transformants compared to those by conventional methods.

Heterologous protein productions using YEp, YCp, and YIp types of yeast expression vectors with ADH1 or GAPDH promoter by the cold culture method showed effective productivity of about 2-fold compared to those by the conventional method of culture at 30°C. The high level production of heterologous protein by this method was not specific to the S. cerevisiae strains examined.     

鲈鱼生长激素在甲醇酵母中的胞内表达

甲醇酵母pichia pastoris是一种理想的真核蛋白高水平表达系统.将鲈鱼(Lateolabrax japonicus)生长激素基因克隆到酵母整合型质粒载体pHIL-D2,经转化his4缺陷型酵母GS115,用PCR方法筛选阳性转化子,并用斑点印迹法筛选多拷贝转化子,经甲醇诱导表达,SDS-PAGE和蛋白质印迹杂交结果证实了表达产物为重组的鲈鱼生长激素.     

The direct repeat sequence upstream of Bacillus chitinase genes is cis-acting elements that negatively regulate heterologous expression in E. coli

To explore the influence of the direct repeat sequence (DRS) in Bacillus chitinase genes on heterogonous expression in Escherichia coli, we cloned and sequenced the entire open reading frame (ORF) and upstream sequences of the chitinase B (chiB) and chitinase MY75 (chiMY75) from Bacillus thuringiensis and Bacillus licheniformis. A pair of 8-bp DRS was found upstream of each chi gene. Chi ORFs with a series of truncated DRS were cloned and transformed into E. coli XL-Blue. The activity of the transformants without the DRS were significantly higher in chitinase assays than transformants containing the DRS. SDS-PAGE showed that part and full deletion of the DRS increased chi gene expression by approximately 1.7 and 3.8-fold, respectively. Northern blotting revealed deletion of the DRS regions increased chiB and chiMY75 mRNA expression. Specific binding of DNA-binding factors in the E. coli cell lyaste was observed to both the chiB and chiMY75 promoter regions and DRS elements. This is the first investigation to demonstrate that heterologous expression of Bacillus chi genes in E. coli is negatively regulated by their upstream DRS regions, which act as cis-acting elements.     

Transgene inactivation inPetunia hybrida is influenced by the properties of the foreign gene

Petunia mutant RL01 was transformed with maizeA1 and gerberagdfr cDNAs, which both encode dihydroflavonol-4-reductase (DFR) activity. The sameAgrobacterium vector and the same version of the CaMV 35S promoter were used in both experiments. Transformation with the cDNAs resulted in production of pelargonidin pigments in the transformants. However, theA1 andgdfr transformants showed clearly different phenotypes. The flowers of the primaryA1 transformants were pale and showed variability in pigmentation during their growth, while the flowers of thegdfr transformants showed intense and highly stable coloration. The color difference in the primary transformants was reflected in the expression levels of the transgenes as well as in the levels of anthocyanin pigment. As previously reported by others, the instability in pigmentation in theA1 transformants was more often detected in clones with multiple copies of the transgene and was associated with methylation of the 35S promoter and of the transgene cDNA itself. In thegdfr transformants, the most intense pigmentation was observed in plants with multiple transgenes in their genome. Only rarely was partial methylation of the 35S promoter detected, while thegdfr cDNA always remained in an unmethylated state. We conclude that the properties of the transgene itself strongly influence the inactivation process. The dicotyledonousgdfr cDNA with a lower GC content and fewer possible methylation sites is more compatible the genomic organization of petunia and this prevents it being recognized as a foreign gene and hence silenced by methylation.     

<Emphasis Type="Italic">Aeromonas caviae</Emphasis> CB101 Contains Four Chitinases Encoded by a Single Gene <Emphasis Type="Italic">chi1</Emphasis>

Aeromonas caviae CB101 secretes four chitinases (around 92, 82, 70, and 55 kDa) into the culture supernatant. A chitinase gene chi1 (92 kDa) was previously studied. To identify the genes encoding the remaining three chitinases, a cosmid library of CB101 was constructed to screen for putative chitinase genes. Nine cosmid clones were shown to contain a chitinase gene on chitin plates. Surprisingly, all the positive clones contained chi1. In parallel, we purified the 55-kDa chitinase (Chi55) from the CB101 culture supernatant by continuous DEAE-Sepharose and Mono-Q anion exchange chromatography. The N-terminal amino acid sequence of the purified chitinase exactly matched the N-terminal sequence of mature Chi1, indicating that the purified chitinase (Chi55) is a truncated form of Chi1. The N- and C-terminal domains of chi1 were cloned, expressed, and purified, separately. Western blots using anti-sera to the N- and C-terminal domains of chi1 on the chitinases of CB101 showed that the four chitinases in the culture supernatant are either chi1 or C-terminal truncations of Chi1. In addition, the CB101 chi1 null mutant showed no chitinolytic activity, while CB101 chi1 null mutant complemented by pUC19chi1 containing chi1 showed all four chitinases in gel activity assay. These data indicated that all four chitinases secreted by CB101 in the culture supernatant are the product of one chitinase gene chi1.     

Transformation and laccase mutant isolation in Coprinus congregatus by restriction enzyme-mediated integration

Coprinus congregatus did not show any growth on a minimal medium in the presence of phosphinothricin which inhibited glutamine synthetase. Genetic transformation to phosphinothricin resistance in C. congregatus was carried out successfully by restriction enzyme-mediated integration. The procedure was improved to yield 550 transformants per μg of DNA, and three laccase mutants were generated. The vector pBARGEM 7-1 which had the phosphinothricin resistance gene was detected in the restriction enzyme fragments of chromosomal DNA from the transformants by Southern hybridization. Transformants showed identical electrophoretic banding patterns but CL1430b had a faster moving band when analyzed by native PAGE.     

Transgenic tomato plants expressing a wheat endochitinase gene demonstrate enhanced resistance to <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">lycopersici</Emphasis>

Various chitinases have been shown to inhibit the growth of fungal pathogens in in vitro as well as in planta conditions. chi194, a wheat chitinases gene encoding a 33-kDa chitinase protein, was overexpressed in tomato plants (cv. Pusa Ruby) under the control of maize ubiquitin 1 promoter. The integration of transgene in tomato plants was confirmed with polymerase chain reaction (PCR) and Southern blot analysis. The inheritance of the transgene in T₁ and T₂ generations were shown by molecular analysis and the hygromycin sensitivity test. The broad range of chitinase activity was observed among the transgenic lines in T₀ and a similar range was retained in the T₁ and T₂ generations. Most importantly, the transgenic tomato lines with high chitinase activity were found to be highly resistant to the fungal pathogen Fusarium oxysporum f. sp. lycopersici. Thus, the results demonstrated that the expression of the wheat endochitinase chi194 in tomato plants confers resistance against Fusarium wilt disease caused by the fungal pathogen Fusarium oxysporum f. sp. lycopersici.     

Efficient transformation of the medicinal mushroomGanoderma lucidum

Ganoderma lucidum is a well-known and important medicinal mushroom, but its genetic modification has not been reported. We developed an efficient procedure for isolation and regeneration of protoplasts fromG. lucidum. To construct a vector for high-level expression of heterologous genes inG. lucidum, the 1.4-kb regulatory region of the glyceraldehyde-3-phosphate dehydrogenase gene (GPD) was isolated from the genomic DNA ofLentinus edodes, and theGPD promoter was fused to the β-glucuronidase (GUS) and bialaphos resistance (bar) genes. Using the resulting construct, p301-bG1, an efficient transformation system based on electroporation was established forG. lucidum. GUS expression was observed among transformants conferring bialaphos resistance, indicating that theL. edodes GPD promoter can be used for expression of exogenous genes inG. lucidum. We also studied green fluorescent protein (GFP) as another reporter for transformation ofG. lucidum. TheL. edodes GPD promoter was fused respectively to theGFP andbar genes, and the resulting construct, p301-bg, was introduced intoG. lucidum. StableGFP expression in transformants was detectable by fluorescence microscopy, suggesting the suitability ofGFP as a reporter system in transformation of this mushroom. This is the first report of an efficient transformation system forG. lucidum using different reporters, paving the way for genetic modification of this famous medicinal mushroom.     

Thermostability and xylan-hydrolyzing property of endoxylanase expressed in yeast Saccharomyces cerevisiae

The endoxylanase gene (xynB, GeneBank access code U51675), including its signal sequence, from Bacillus spp. was amplified and connected in frame downstream of yeast ADH1 promoter and then the resulting plasmid, pAEDX-1, was introduced into Saccharomyces cerevisiae. When the yeast transformants were grown on YPD medium, the majority of endoxylanase activity was detected in the extracellular culture medium, indicating that the signal peptide of Bacillus endoxylanase functioned well in yeast. In the batch cultivation of yeast transformants, the total expression level of endoxylanase and secretion efficiency were measured to be about 9.8 U/mL and 66.2%, respectively. The extracellular endoxylanase expressed in yeast showed an enhanced thermal stability due to the N-linked glycosylation. Through the hydrolysis of birchwood xylan with the endoxylanase, it was found that xylobiose and xylotriose were produced as major products with equimolar ratio.     

Development of strains of the thermotolerant yeast Hansenula polymorpha capable of alcoholic fermentation of starch and xylan

The thermotolerant yeast Hansenula polymorpha ferments glucose and xylose to ethanol at high temperatures. However, H. polymorpha cannot utilize starchy materials or xylans. Heterologous amylolytic and xylanolytic enzymes have to be expressed in this yeast to provide for utilization and growth on starch and xylan. Genes SWA2 and GAM1 from the yeast Schwanniomyces occidentalis, encoding α-amylase and glucoamylase, respectively, were expressed in H. polymorpha. The expression was achieved by integration of the SWA2 and GAM1 genes under the strong constitutive promoter of the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase gene (HpGAP) into H. polymorpha genome. Resulting transformants acquired the ability to grow on a minimal medium containing soluble starch as a sole carbon source. Ethanol production at high-temperature fermentation from starch by the recombinant strains was up to 10 g/L. The XYN2 gene encoding endoxylanase of the fungus Trichoderma reseei was expressed in H. polymorpha. Co-expression of xlnD gene coding for β-xylosidase of the fungus Aspergillus niger and the XYN2 gene in H. polymorpha was achieved by integration of these genes under control of the HpGAP promoter. Resulting transformants were capable of growth and alcoholic fermentation on a minimal medium supplemented with birchwood xylan as a sole carbon source at 48 °C.     

Inhibition of Neurospora crassa Growth by a Glucan Synthase-1 Antisense Construct

We have used the filamentous fungus, Neurospora crassa, as a model system to test the concept that antisense targeting of the cell-wall assembly enzyme, (1,3)β-glucan synthase [E.C. 2.4.1.34; UDP glucose: 1,3-β-D-glucan 3-β-D-glucosyltransferase], leads to a corresponding decrease in growth of the organism. Previously, our laboratory isolated a gene (glucan synthase-1, gs-1) that is required for (1,3)β-glucan synthase activity. Wild-type cells were transformed with DNA vectors encoding various RNAs complementary to the gs-1 messenger RNA (antisense RNA) cloned downstream from an inducible promoter (quinic acid-2 [qa-2p]). Stable transformants, expressing a partially inverted antisense message of gs-1 (pMYX107), exhibited dramatic reduction in growth compared with empty vector controls. Hyphal measurements of these transformants grown on race tubes indicated that all of the transformants showed various degrees of inhibition. Microscopic observations of transformants revealed shorter hyphal lengths when grown under conditions expressing antisense. Further characterization revealed that the specific activities of (1,3)β-glucan synthase were decreased by as much as 63% relative to empty vector controls. Together, these observations suggest that antisense against (1,3)β-glucan synthase led to a reduction in enzyme levels that resulted in altered cell-wall morphology and inhibition of growth. It is possible that antisense oligonucleotides against gs-1 may be useful antifungal agents. Received: 20 September 1996 / Accepted: 1 November 1996