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Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

Wang ZL, Ying SH, Feng MG - Microb Biotechnol (2012)

Bottom Line: Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein).Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi.Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

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Overexpression of vip3Aa1 in B. bassiana strains engineered under Phyd1-t1 control. a. Concatenation of expression elements in binary plasmid constructed for transformation.b. PCR detection for the presence of vip3Aa1 in 12 transformants (lanes 1−12). P, BbV28; C, wild-type (Bb2860).c. Relative transcript levels of vip3Aa1 in 4 day colonies of eight positive transformants (under Phyd1-t1 control) versus PgpdA-controlled BbV28 in qRT-PCR. d and e. Relative expression levels of vip3Aa1 in the mycelia and conidia of five selected transformants versus BbV28 in elisa respectively. f. Western blots of mycelial (m) and conidial (c) extracts of Bb2860 (left), BbHV8 (middle) and BbV28 (right) probed by the polyclonal antibody of vip3Aa1. g–i. Immunogold localization of vip3Aa1 molecules expressed in the conidia of Bb2860, BbV28 and BbHV8 respectively. Note that 10 nm colloidal gold particles labelled with the polyclonal antibody and goat anti-rabbit IgG antibody are much denser in BbHV8 (i) than in BbV28 (h) but absent in Bb2860 (g).
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fig02: Overexpression of vip3Aa1 in B. bassiana strains engineered under Phyd1-t1 control. a. Concatenation of expression elements in binary plasmid constructed for transformation.b. PCR detection for the presence of vip3Aa1 in 12 transformants (lanes 1−12). P, BbV28; C, wild-type (Bb2860).c. Relative transcript levels of vip3Aa1 in 4 day colonies of eight positive transformants (under Phyd1-t1 control) versus PgpdA-controlled BbV28 in qRT-PCR. d and e. Relative expression levels of vip3Aa1 in the mycelia and conidia of five selected transformants versus BbV28 in elisa respectively. f. Western blots of mycelial (m) and conidial (c) extracts of Bb2860 (left), BbHV8 (middle) and BbV28 (right) probed by the polyclonal antibody of vip3Aa1. g–i. Immunogold localization of vip3Aa1 molecules expressed in the conidia of Bb2860, BbV28 and BbHV8 respectively. Note that 10 nm colloidal gold particles labelled with the polyclonal antibody and goat anti-rabbit IgG antibody are much denser in BbHV8 (i) than in BbV28 (h) but absent in Bb2860 (g).

Mentions: Interestingly, the eGFP expression under Phyd1-t1 control was barely detected during the first 48 h of incubation of selected transformants on Sabouraud dextrose agar plus yeast extract (SDAY) at 25°C but rapidly increased following this time period. The eGFP-emitted fluorescence under laser confocal microscope was much more intense in conidiogenic cells and conidia than in younger mycelia (Fig. 2b). Mean RFI in the colonies increased from 2.4 on day 2 to 1187 on day 4 and reached a peak of 1842 on day 7 (Fig. 2c), when conidiation was completed. These data indicated that Phyd1-t1 enabled the activation of gene expression specifically during the conidiophore development and conidiation of B. bassiana.


Recognition of a core fragment ofBeauveria bassiana hydrophobin gene promoter (P hyd1) and its special use in improving fungal biocontrol potential.

Wang ZL, Ying SH, Feng MG - Microb Biotechnol (2012)

Overexpression of vip3Aa1 in B. bassiana strains engineered under Phyd1-t1 control. a. Concatenation of expression elements in binary plasmid constructed for transformation.b. PCR detection for the presence of vip3Aa1 in 12 transformants (lanes 1−12). P, BbV28; C, wild-type (Bb2860).c. Relative transcript levels of vip3Aa1 in 4 day colonies of eight positive transformants (under Phyd1-t1 control) versus PgpdA-controlled BbV28 in qRT-PCR. d and e. Relative expression levels of vip3Aa1 in the mycelia and conidia of five selected transformants versus BbV28 in elisa respectively. f. Western blots of mycelial (m) and conidial (c) extracts of Bb2860 (left), BbHV8 (middle) and BbV28 (right) probed by the polyclonal antibody of vip3Aa1. g–i. Immunogold localization of vip3Aa1 molecules expressed in the conidia of Bb2860, BbV28 and BbHV8 respectively. Note that 10 nm colloidal gold particles labelled with the polyclonal antibody and goat anti-rabbit IgG antibody are much denser in BbHV8 (i) than in BbV28 (h) but absent in Bb2860 (g).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3815382&req=5

fig02: Overexpression of vip3Aa1 in B. bassiana strains engineered under Phyd1-t1 control. a. Concatenation of expression elements in binary plasmid constructed for transformation.b. PCR detection for the presence of vip3Aa1 in 12 transformants (lanes 1−12). P, BbV28; C, wild-type (Bb2860).c. Relative transcript levels of vip3Aa1 in 4 day colonies of eight positive transformants (under Phyd1-t1 control) versus PgpdA-controlled BbV28 in qRT-PCR. d and e. Relative expression levels of vip3Aa1 in the mycelia and conidia of five selected transformants versus BbV28 in elisa respectively. f. Western blots of mycelial (m) and conidial (c) extracts of Bb2860 (left), BbHV8 (middle) and BbV28 (right) probed by the polyclonal antibody of vip3Aa1. g–i. Immunogold localization of vip3Aa1 molecules expressed in the conidia of Bb2860, BbV28 and BbHV8 respectively. Note that 10 nm colloidal gold particles labelled with the polyclonal antibody and goat anti-rabbit IgG antibody are much denser in BbHV8 (i) than in BbV28 (h) but absent in Bb2860 (g).
Mentions: Interestingly, the eGFP expression under Phyd1-t1 control was barely detected during the first 48 h of incubation of selected transformants on Sabouraud dextrose agar plus yeast extract (SDAY) at 25°C but rapidly increased following this time period. The eGFP-emitted fluorescence under laser confocal microscope was much more intense in conidiogenic cells and conidia than in younger mycelia (Fig. 2b). Mean RFI in the colonies increased from 2.4 on day 2 to 1187 on day 4 and reached a peak of 1842 on day 7 (Fig. 2c), when conidiation was completed. These data indicated that Phyd1-t1 enabled the activation of gene expression specifically during the conidiophore development and conidiation of B. bassiana.

Bottom Line: Further truncating P hyd1-t1 to 1179, 991 and 791 bp or mutating one of the binding domains of three transcription factors in P hyd1-t1 reduced significantly the expression of eGFP (enhanced green fluorescence protein).Therefore, P hyd1-t1 was used to integrate a bacterium-derived, insect midgut-specific toxin (vip3Aa1) gene into B. bassiana, yielding a transgenic strain (BbHV8) expressing 9.8-fold more toxin molecules in conidia than a counterpart strain (BbV28) expressing the toxin under the control of P gpdA, a promoter widely used for gene expression in fungi.Conclusively, P hyd1-t1 is a useful tool for enhancing beneficial protein expression, such as vip3Aa1, in fungal conidia, which are the active ingredients of mycoinsecticides.

View Article: PubMed Central - PubMed

Affiliation: Institute of Microbiology, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

Show MeSH
Related in: MedlinePlus