Liqiong Guo

Highly efficient Agrobacterium-mediated transformation of Volvariella volvacea.

Agrobacterium tumefaciens-mediated transformation (ATMT) was successfully applied to the edible straw mushroom, Volvariella volvacea. Mycelium pellets were transformed to cold stress resistance using the afp gene as both a selective marker and a reporter gene, under the control of a heterologous Lentinula edodes gpd promoter. The efficiency of transformation is over 100 times higher than that previously reported in V. volvacea. Stable integration of the afp gene with 1-4 copy numbers was confirmed in all 10 randomly selected transgenic events by Southern blot analysis. The mitotic stability of the transformants was demonstrated after five successive transfers on PDA medium without selection pressure and the PCR analysis of basidiospores harvested from transformants.

Expression of multi-functional cellulase gene mfc in Coprinus cinereus under control of different basidiomycete promoters

Multi-functional cellulase gene mfc was expressed in Coprinus cinereus under naturally non-inductive conditions using three heterologous promoters. Endo-beta-1,4-glucanase expression was achieved in solid and liquid media with promoter sequences from the Lentinula edodesgpd gene, the Flammulina velutipes gpd gene and the Volvariella volvaceagpd gene. As measured by enzyme activity in liquid cultures, a 613-bp gpd promoter fragment from L. edodes was most efficient, followed by a 752-bp gpd fragment from F. velutipes. The V. volvacea gpd promoter sequence was less active, in comparison. Irrespective of the promoter used, enzymatic activities increase 34-fold for highly active transformants and 29-fold for less active one by using cellulase-inducing medium. The highest activities of endo-beta-1,4-glucanase (34.234 U/ml) and endo-beta-1,4-xylanase (263.695 U/ml) were reached by using the L. edodesgpd promoter.

Molecular cloning and characterization of a novel laccase gene from a white‐rot fungus Polyporus grammocephalus TR16 and expression in Pichia pastoris

Aims:  To isolate a novel laccase gene from white‐rot fungus Polyporus grammocephalus TR16 and heterologous expression in Pichia pastoris. The characteristics of the heterologously expressed laccase are also studied.

Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea.

Aims:  FIP‐gsi, a fungal immunomodulatory protein found in Ganoderma sinense, has antitumour, anti‐allergy and immunomodulatory activities and is regulated by the fip‐gsi gene. In this study, we aimed to express the fip‐gsi gene from G. sinense in Coprinopsis cinerea to increase yield of FIPs‐gsi.

Improvement in fruiting body yield by introduction of the Ampullaria crossean multi-functional cellulase gene into Volvariella volvacea.

The multi-functional cellulase gene, mfc, from Ampullaria crossean was transformed into Volvariella volvacea by PEG-mediated protoplast transformation to improve the biological efficiency and fruiting body yield. PCR and Southern blotting indicated that mfc was integrated into the genomes of four transformants. In laboratory and large scale cultivation experiments, the average biological efficiency of the transformants was 18.39+/-1.27% and 27.84+/-3.21%, respectively, considerably higher than the corresponding values for untransformed controls of 12.69+/-1.31% and 20.63+/-2.59%. This is the first report of an improvement in biological efficiency and fruiting body yield of V. volvacea through transgenesis.

A rapid pulsed-field gel electrophoresis method of genotyping Haemophilus parasuis isolates.

Aims:  To develop a modified pulsed‐field gel electrophoresis (PFGE) method for characterizing Haemophilus parasuis isolates.