Project Title

Detection of Chitinase and β-1, 3 Glucanase Genes Against Aspergillus flavus in Transformed Peanuts

Presenters

Academic department under which the project should be listed

CSM - Molecular and Cellular Biology

Faculty Sponsor Name

Dr. Premila Achar

Additional Faculty

Detection of Chitinase and β-1, 3 Glucanase Genes Against Aspergillus flavus in Transformed Peanuts Solarin, R., Bokanya, G., Lazo-Gonzalez, R., Achar, P.N.1 1Dept. of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA Infection of peanut crops by Aspergillus flavus and A. parasiticus is a serious problem in Georgia. Specifically, Aspergillus flavus and A. parasiticus produce aflatoxins, which are among the most carcinogenic and naturally occurring compounds known to pose significant health risks to human and animals. There is no direct action peanut farmers can take to control fungi that produce aflatoxin in peanut seeds. Transgenic approaches are being undertaken to prevent invasion by Aspergillus fungi or to prevent biosynthesis of aflatoxin. The hydrolytic enzymes, rice based chitinase and β-1, 3 glucanase, have been established to be active defense mechanisms against various pathogens, especially fungal diseases, with particular reference to A. flavus. The goal of this project was to transform peanut plants with chitinase and glucanase genes against A. flavus, via Agrobacterium mediated transformation. Somatic embryos were transformed by co-cultivating with Agrobacterium tumefaciens plasmid constructs (pCAMBARGlu 289 & pCAMBAR Chi11, Dr. Muthukrishnan, Kansas State Univ). Specific primers were used to confirm successful gene inserts into transgenic peanuts by Polymerase Chain Reaction (PCR). While the Chitinase gene, was detected at ~700bp, the Glucanase was amplified at ~1200bp in the genetically modified (GM) peanuts. The quantification of these pathogen-related (PR) enzymes was done using a GUS histochemical assay. The GUS histochemical assay confirmed successful gene insert into the transgenic explants, however, Chitinase was not stably inserted. Characterization of expressed β-1 3 Glucanase gene was done by SDS-PAGE and Western Blot analysis. Estimation of total protein in GM peanuts was done by standard Bradford method. Our present study successfully standardized somatic embryogenesis and regeneration protocol in peanuts. The successful transformation led to the production of 50% or more of transgenic plants as evidenced by the GUS assay. While further tests will merely confirm the successful expression of proteins, we conclude that the transgenic peanut lines possessing fungal resistance genes may offer an alternative to traditional breeding and use of synthetic fungicides, in managing Aspergillus species.

Abstract (300 words maximum)

Detection of Chitinase and β-1, 3 Glucanase Genes Against Aspergillus flavus in Transformed Peanuts

Solarin, R., Bokanya, G., Lazo-Gonzalez, R., Achar, P.N.1

1Dept. of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA

Infection of peanut crops by Aspergillus flavus and A. parasiticus is a serious problem in Georgia. Specifically, Aspergillus flavus and A. parasiticus produce aflatoxins, which are among the most carcinogenic and naturally occurring compounds known to pose significant health risks to human and animals. There is no direct action peanut farmers can take to control fungi that produce aflatoxin in peanut seeds. Transgenic approaches are being undertaken to prevent invasion by Aspergillus fungi or to prevent biosynthesis of aflatoxin. The hydrolytic enzymes, rice based chitinase and β-1, 3 glucanase, have been established to be active defense mechanisms against various pathogens, especially fungal diseases, with particular reference to A. flavus. The goal of this project was to transform peanut plants with chitinase and glucanase genes against A. flavus, via Agrobacterium mediated transformation. Somatic embryos were transformed by co-cultivating with Agrobacterium tumefaciens plasmid constructs (pCAMBARGlu 289 & pCAMBAR Chi11, Dr. Muthukrishnan, Kansas State Univ). Specific primers were used to confirm successful gene inserts into transgenic peanuts by Polymerase Chain Reaction (PCR). While the Chitinase gene, was detected at ~700bp, the Glucanase was amplified at ~1200bp in the genetically modified (GM) peanuts. The quantification of these pathogen-related (PR) enzymes was done using a GUS histochemical assay. The GUS histochemical assay confirmed successful gene insert into the transgenic explants, however, Chitinase was not stably inserted. Characterization of expressed β-1 3 Glucanase gene was done by SDS-PAGE and Western Blot analysis. Estimation of total protein in GM peanuts was done by standard Bradford method. Our present study successfully standardized somatic embryogenesis and regeneration protocol in peanuts. The successful transformation led to the production of 50% or more of transgenic plants as evidenced by the GUS assay. While further tests will merely confirm the successful expression of proteins, we conclude that the transgenic peanut lines possessing fungal resistance genes may offer an alternative to traditional breeding and use of synthetic fungicides, in managing Aspergillus species.

Project Type

Poster

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Detection of Chitinase and β-1, 3 Glucanase Genes Against Aspergillus flavus in Transformed Peanuts

Detection of Chitinase and β-1, 3 Glucanase Genes Against Aspergillus flavus in Transformed Peanuts

Solarin, R., Bokanya, G., Lazo-Gonzalez, R., Achar, P.N.1

1Dept. of Molecular and Cellular Biology, Kennesaw State University, Kennesaw, GA

Infection of peanut crops by Aspergillus flavus and A. parasiticus is a serious problem in Georgia. Specifically, Aspergillus flavus and A. parasiticus produce aflatoxins, which are among the most carcinogenic and naturally occurring compounds known to pose significant health risks to human and animals. There is no direct action peanut farmers can take to control fungi that produce aflatoxin in peanut seeds. Transgenic approaches are being undertaken to prevent invasion by Aspergillus fungi or to prevent biosynthesis of aflatoxin. The hydrolytic enzymes, rice based chitinase and β-1, 3 glucanase, have been established to be active defense mechanisms against various pathogens, especially fungal diseases, with particular reference to A. flavus. The goal of this project was to transform peanut plants with chitinase and glucanase genes against A. flavus, via Agrobacterium mediated transformation. Somatic embryos were transformed by co-cultivating with Agrobacterium tumefaciens plasmid constructs (pCAMBARGlu 289 & pCAMBAR Chi11, Dr. Muthukrishnan, Kansas State Univ). Specific primers were used to confirm successful gene inserts into transgenic peanuts by Polymerase Chain Reaction (PCR). While the Chitinase gene, was detected at ~700bp, the Glucanase was amplified at ~1200bp in the genetically modified (GM) peanuts. The quantification of these pathogen-related (PR) enzymes was done using a GUS histochemical assay. The GUS histochemical assay confirmed successful gene insert into the transgenic explants, however, Chitinase was not stably inserted. Characterization of expressed β-1 3 Glucanase gene was done by SDS-PAGE and Western Blot analysis. Estimation of total protein in GM peanuts was done by standard Bradford method. Our present study successfully standardized somatic embryogenesis and regeneration protocol in peanuts. The successful transformation led to the production of 50% or more of transgenic plants as evidenced by the GUS assay. While further tests will merely confirm the successful expression of proteins, we conclude that the transgenic peanut lines possessing fungal resistance genes may offer an alternative to traditional breeding and use of synthetic fungicides, in managing Aspergillus species.