Construction and Preliminary Evaluation of an Aspergillus flavus Reporter Gene Construct as a Potential Tool for Screening Aflatoxin Resistance

2003 ◽  
Vol 66 (10) ◽  
pp. 1927-1931 ◽  
Author(s):  
ROBERT L. BROWN ◽  
CARMEN S. BROWN-JENCO ◽  
DEEPAK BHATNAGAR ◽  
GARY A. PAYNE
Keyword(s):  
Reporter Gene ◽  
Aspergillus Flavus ◽  
Fungal Growth ◽  
Gus Expression ◽  
Alternative Methods ◽  
Preliminary Evaluation ◽  
Gene Construct ◽  
Reporter Gene Construct ◽  
Aflatoxin Accumulation ◽  
Maize Kernels

Effective preharvest strategies to eliminate aflatoxin accumulation in crops are not presently available. The molecular biology of aflatoxin biosynthesis has been extensively studied, and genetic and molecular tools such as reporter gene systems for the measurement of fungal growth have been developed. A reporter construct containing the Aspergillus flavus β-tubulin gene promoter fused to Escherichia coli β-glucuronidase (GUS) has been shown to be a reliable tool for the indirect measurement of fungal growth in maize kernels. Since cost-saving alternative methods for the direct measurement of aflatoxin levels are needed to facilitate more widespread field and laboratory screening of maize lines, a new reporter gene construct involving the promoter region of the omtA gene of the aflatoxin biosynthetic pathway was constructed and tested. Expression of GUS activity by this construct (omtA::GUS) was correlated with aflatoxin accumulation in culture. In the fungal transformant GAP26-1, which harbors this construct, aflatoxin production and GUS expression on sucrose-containing medium showed the same temporal pattern of toxin induction. Furthermore, GUS expression by GAP26-1 was shown to be associated with aflatoxin accumulation in maize kernels inoculated with this strain. Our results suggest that this and other reporter gene pathway promoter constructs may provide superior alternatives to direct aflatoxin quantification with respect to time, labor, and materials for the screening of maize lines for resistance to aflatoxin accumulation.

Growth of an Aspergillus flavus Transformant Expressing Escherichia coli β-Glucuronidase in Maize Kernels Resistant to Aflatoxin Production

1997 ◽  
Vol 60 (1) ◽  
pp. 84-87 ◽  
Author(s):  
ROBERT L. BROWN ◽  
THOMAS E. CLEVELAND ◽  
GARY A. PAYNE ◽  
CHARLES P. WOLOSHUK ◽  
DONALD G. WHITE
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Flavus ◽  
Aflatoxin B1 ◽  
Gene Promoter ◽  
Fungal Growth ◽  
Aflatoxin Production ◽  
Glucuronidase Activity ◽  
Glucuronidase Reporter ◽  
Aflatoxin Accumulation ◽  
Maize Kernels

Kernels of a maize inbred that demonstrated resistance to aflatoxin production in previous studies were inoculated with an Aspergillus flavus strain containing the Escherichia coli β-d-glucuronidase reporter gene linked to a β-tubulin gene promoter and assessed for both fungal growth and aflatoxin accumulation. Prior to inoculation, kernels were pin-wounded through the pericarp to the endosperm, pin-wounded in the embryo region, or left unwounded. After 7 days incubation with the fungus, β-glucuronidase activity (fungal growth) in the kernels was quantified using a fluorogenic assay and aflatoxin B1 content of the same kernels was analyzed. Kernels of a susceptible inbred, similarly treated, served as controls. Results indicate a positive relationship between aflatoxin levels and the amount of fungal growth. However, resistant kernels wounded through the pericarp to the endosperm before inoculation supported an increase in aflatoxin B1 over levels observed in nonwounded kernels, without an increase in fungal growth. Wounding kernels of the resistant inbred through the embryo resulted in both the greatest fungal growth and the highest levels of aflatoxin B1 for this genotype. Maintenance of resistance to aflatoxin B1 in endosperm-wounded kernels may be due to the action of a mechanism which limits fungal access to the kernel embryo.

scholarly journals A type I collagen reporter gene construct for protein engineering studies. Functional equivalence of transfected reporter COL1A1 and endogenous gene products during biosynthesis and in vitro extracellular matrix accumulation

1993 ◽  
Vol 293 (2) ◽  
pp. 387-394 ◽  
Author(s):  
S R Lamandé ◽  
J F Bateman
Keyword(s):  
Extracellular Matrix ◽  
Reporter Gene ◽  
Type I Collagen ◽  
Type I ◽  
Wild Type ◽  
Reporter Construct ◽  
Reporter Protein ◽  
Gene Construct ◽  
Reporter Gene Construct

A type I collagen reporter gene construct, designed to facilitate detailed analysis of the consequences of introduced structural and regulatory mutations on collagen biosynthesis and participation in the extracellular matrix, was produced by site-directed mutagenesis of the mouse COL1A1 gene. The reporter construct, pWTCI-Ile822, carried a single base change which converted the codon for amino acid 822 of the triple helix from methionine to isoleucine. This change allowed the reporter protein, [Ile822]alpha 1(I), to be distinguished from the wild-type alpha 1(I), and quantified, by its altered CNBr cleavage pattern. In mouse Mov13 cells, which synthesize no endogenous pro alpha 1(I), reporter chains associated with endogenous pro alpha 2(I), formed pepsin-stable triple helices and were secreted efficiently from the cell. The thermal stability of wild-type molecules and molecules containing the reporter [Ile822]alpha 1(I) chains was identical. The biosynthetic characteristics of wild-type and reporter chains were directly compared in stably transfected 3T6 cells. These cells did not make a distinction between reporter and endogenous alpha 1(I) chains, which were secreted from the cells at the same rate and were processed and deposited into the 3T6 cell in vitro accumulated extracellular matrix with equal efficiency. These data demonstrate that the helical sequence alteration in the reporter protein is functionally neutral and that the reporter construct, pWTCI-Ile822, is a suitable vector for the analysis of the biochemical effects of site-directed mutations in the putative COL1A1 functional domains.

Living Maize Embryo Influences Accumulation of Aflatoxin in Maize Kernels

1993 ◽  
Vol 56 (11) ◽  
pp. 967-971 ◽  
Author(s):  
ROBERT L. BROWN ◽  
PETER J. COTTY ◽  
THOMAS E. CLEVELAND ◽  
NEIL W. WIDSTROM
Keyword(s):  
Aspergillus Flavus ◽  
Fungal Growth ◽  
Aflatoxin Contamination ◽  
Population Variation ◽  
Maize Populations ◽  
Susceptible Control ◽  
Maize Embryo ◽  
Metabolic Activities ◽  
Maize Kernels ◽  
Two Populations

Kernels from two maize populations, MAS:gk and MAS:pw,nf, showed significant postharvest resistance to aflatoxin contamination by Aspergillus flavus but showed no significant inter-population variation for this resistance. Growth of A. flavus on both populations was significantly less than on susceptible control lines. Kernels from the resistant populations retained resistance when wounded through the pericarp prior to inoculation with A. flavus, despite the fact that the exposed endosperm supported good fungal growth. Kernels from these populations also retained resistance when they were acetone washed before inoculation. Resistance to aflatoxin contamination was lost in kernels that were autoclaved, crushed, or embryo wounded. All assays were incubated under conditions favorable to kernel germination. Results suggest that postharvest resistance to aflatoxin contamination in these two populations is related to metabolic activities of the living com embryo.

Tissue and cell specific expression of a renin promoter-reporter gene construct in transgenic mice

1990 ◽  
Vol 170 (1) ◽  
pp. 344-350 ◽  
Author(s):  
Curt D. Sigmund ◽  
Craig A. Jones ◽  
John R. Fabian ◽  
John J. Mullins ◽  
Kenneth W. Gross
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Specific Expression ◽  
Gene Construct ◽  
Reporter Gene Construct

scholarly journals Reporter gene construct containing 1.4-kB ?1-proteinase inhibitor promoter confers expression in the cornea of transgenic mice

2001 ◽  
Vol 266 (1) ◽  
pp. 5-9
Author(s):  
Jun Ueda ◽  
Yuhong Li ◽  
Meilan Stephanie Goh ◽  
Yasuhiro Maruyama ◽  
Joel Sugar ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Reporter Gene ◽  
Proteinase Inhibitor ◽  
Gene Construct ◽  
Reporter Gene Construct

An inhibitor of aflatoxin biosynthesis in developing cottonseed

1988 ◽  
Vol 66 (5) ◽  
pp. 998-1002 ◽  
Author(s):  
Susan P. McCormick ◽  
Deepak Bhatnagar ◽  
Wilton R. Goynes ◽  
Louise S. Lee
Keyword(s):  
Growth Inhibition ◽  
Molecular Mass ◽  
Aspergillus Flavus ◽  
Anion Exchanger ◽  
Inhibitory Activity ◽  
Fungal Growth ◽  
Aflatoxin Biosynthesis ◽  
Seed Moisture ◽  
Aflatoxin Accumulation ◽  
Fungal Growth Inhibition

A factor present in the coats of young developing cottonseed (20–25 days old) but absent from older developing seeds (35–40 days old) significantly inhibited (> 85%) aflatoxin formation by Aspergillus flavus without affecting fungal growth. Inhibition was independent of levels of seed moisture, phenolics, or tannins. The inhibitory factor was nondialyzable (molecular mass > 8 kDa); the inhibition of aflatoxin accumulation was dependent on its concentration. The inhibitor was probably an anionic protein since it bound to an anion exchanger and was eluted at 0.15 M salt. The inhibitor did not exhibit a peroxidase-like activity, even though it was observed that the factor was stable at temperatures below 70 °C and that sugar moieties were associated with the inhibitory property. The inhibitory activity was not similar to that of a serine protease.

scholarly journals A Chitinase from Tex6 Maize Kernels Inhibits Growth of Aspergillus flavus

2004 ◽  
Vol 94 (1) ◽  
pp. 82-87 ◽  
Author(s):  
Kenneth G. Moore ◽  
Michael S. Price ◽  
Rebecca S. Boston ◽  
Arthur K. Weissinger ◽  
Gary A. Payne
Keyword(s):  
Aspergillus Flavus ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chitinase Activity ◽  
Peptide Sequence ◽  
Inhibitory Protein ◽  
Maize Genotype ◽  
Aflatoxin Accumulation ◽  
Maize Kernels

The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an Mr of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45°C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 μg/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.

Sign in / Sign up

Export Citation Format

Share Document