Promotion of the growth and plant biomass degrading enzymes production in solid-state cultures of Lentinula edodes expressing Vitreoscilla hemoglobin gene.

Vitreoscilla hemoglobin (VHb), encoded by the Vitreoscilla hemoglobin gene (vgb), is highly effective at binding oxygen and delivering it to both prokaryotes and eukaryotes under hypoxic conditions. In this study, we introduced the vgb gene into shiitake mushrooms, and the mycelia of the transformatants grew faster. In particular, they spread into the solid substrate located in the lower part of the test tubes and bags where the oxygen was hypoxic and produced more ß-glucan and plant biomass degrading enzymes compared to the original strain. The maximum growth rate of the transformants was 8.5%-15.9% higher than that of the original strain on sawdust-based cultures in plastic bags. The laccase and amylase activities were 17.7%-40.3% and 16.7%-37.9% higher than that of the original strain, respectively. In addition, the ß-glucan contents of the transformant mycelia from the submerged fermentation were 12.9%-24.0% higher than that of the original strain. These results reveal that the expression of VHb in mushroom fungi promots the mycelial growth in solid-state cultures under the hypoxic condition as well as enhances ß-glucan and plant biomass degrading enzymes production.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus.

Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13×3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13×3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.

[Comparative study of sensitivity of different dental metal materials].

PURPOSE: To compare the sensitivity of different dental metal materials, in order to provide references for choosing of dental metal materials. METHODS: Patch test was performed on 92 patients wearing dental metal prosthesis. Pearson Chi-square test, corrected Chi-square test and Fisher exact test were used for statistical analysis with SPSS17.0 software package. RESULTS: (1)The sensitivity rates of different metal materials were different. The allergy rate of nickel (Ni) was the highest (22.8%), while the allergy rate of aluminum (Al) was 0. (2)More women were allergic to both palladium (Pd) and nickel (Ni) than men with significant difference (P>0.05). (3)Women with ear piercing were more allergic to nickel (Ni), but there was no significant correlation between ear piercing and nickel allergy (P>0.05).(4)There was cross reaction between nickel(Ni)and palladium (Pd), 83.3% of palladium (Pd) allergy patients were allergic to nickel (Ni), while 47.6% of nickel(Ni) allergy patients were allergic to palladium (Pd). (5)Patch test had a delayed reaction. CONCLUSIONS: Dental metal materials have certain allergies, women are more allergic to both palladium (Pd) and nickel (Ni) than men, with significant difference. Patch test may have a delayed reaction. If necessary, observation for 96 h, 7 days or even longer time, are needed to exclude false positivity. Supported by Research Fund of Science and Technology Committee of Shanghai Municipality (10411950900).

Improving the expression of recombinant pullulanase by increasing mRNA stability in Escherichia coli

Background: Pullulanase production in both wild-type strains and recombinantly engineered strains remains low. The Shine-Dalgarno (SD) sequence and stem-loop structure in the 5' or 3' untranslated region (UTR) are well-known determinants of mRNA stability. This study investigated the effect of mRNA stability on pullulanase heterologous expression. Results: We constructed four DNA fragments, pulA, SD-pulA, pulA-3t, and SD-pulA-3t, which were cloned into the expression vector pHT43 to generate four pullulanase expression plasmids. The DNA fragment pulA was the coding sequence (CDS) of pulA in Klebsiella variicola Z-13. SD-pulA was constructed by the addition of the 5' SD sequence at the 5' UTR of pulA. pulA-3t was constructed by the addition of a 3' stem-loop structure at the 3' UTR of pulA. SD-pulA-3t was constructed by the addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of pulA. The four vectors were transformed into Escherichia coli BL21(DE3). The pulA mRNA transcription of the transformant harboring pHT43-SD-pulA-3t was 338.6%, 34.9%, and 79.9% higher than that of the other three transformants, whereas the fermentation enzyme activities in culture broth and intracellularly were 107.0 and 584.1 times, 1.2 and 2.0 times, and 62.0 and 531.5 times the amount of the other three transformants (pulA, SD-pulA, and pulA-3 t), respectively. Conclusion: The addition of the 5' SD sequence at the 5' UTR and a 3' stem-loop structure at the 3' UTR of the pulA gene is an effective approach to increase pulA gene expression and fermentation enzyme activity.

Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus

Abstract Dikarya is a subkingdom of fungi that includes Ascomycota and Basidiomycota. The gene expression patterns of dikaryon are poorly understood. In this study, we bred a dikaryon DK13 × 3 by mating monokaryons MK13 and MK3, which were from the basidiospores of Pleurotus ostreatus TD300. Using RNA-Seq, we obtained the transcriptomes of the three strains. We found that the total transcript numbers in the transcriptomes of the three strains were all more than ten thousand, and the expression profile in DK13 × 3 was more similar to MK13 than MK3. However, the genes involved in macromolecule utilization, cellular material synthesis, stress-resistance and signal transduction were much more up-regulated in the dikaryon than its constituent monokaryons. All possible modes of differential gene expression, when compared to constituent monokaryons, including the presence/absence variation, and additivity/nonadditivity gene expression in the dikaryon may contribute to heterosis. By sequencing the urease gene poure sequences and mRNA sequences, we identified the monoallelic expression of the poure gene in the dikaryon, and its transcript was from the parental monokaryon MK13. Furthermore, we discovered RNA editing in the poure gene mRNA of the three strains. These results suggest that the gene expression patterns in dikaryons should be similar to that of diploids during vegetative growth.