Home › Microbiology › RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem
Microbiology JoVE (Open Access) Citable · DOI

RNAi-mediated Control of Aflatoxins in Peanut: Method to Analyze Mycotoxin Production and Transgene Expression in the Peanut/Aspergillus Pathosystem

DOI: 10.3791/53398-v
What you'll learn
  • ✓ Prepare and validate transgenic peanut seeds expressing RNAi constructs targeting aflatoxin synthesis
  • ✓ Quantify aflatoxin levels in peanut seeds using analytical chemistry methods
  • ✓ Confirm transgene expression via RT-PCR and correlate with fungal resistance phenotypes
Protocol

We demonstrate a method for the analysis of aflatoxins and transgene expression in peanut seeds that contain RNA-interference signals for silencing aflatoxin-synthesis genes in the fungus Aspergillus flavus. RNAi-mediated control of mycotoxins in plants has not been reported previously.

Difficulty
advanced
Total time
~2–3 weeks (seed preparation, fungal infection, extraction, and analysis cycles)
Model organism
Peanut (Arachis hypogaea) infected with Aspergillus flavus
Biosafety
BSL-2

Steps

1
Prepare transgenic and control peanut seeds

Obtain and organize transgenic peanut seeds containing RNAi constructs and non-transgenic control seeds. Ensure seeds are properly stored and documented before proceeding to infection studies.

▶ 00:41
2
Analyze aflatoxin levels via chromatography

Extract and measure aflatoxin concentrations from peanut seed samples using high-performance liquid chromatography (HPLC) or comparable analytical methods to quantify mycotoxin production differences.

▶ 04:40
3
Confirm transgene expression using RT-PCR

Perform reverse-transcription PCR on peanut seed RNA to verify expression of RNAi constructs targeting aflatoxin-synthesis genes in Aspergillus.

▶ 06:58
4
Correlate RNAi expression with aflatoxin resistance

Compare aflatoxin production levels between transgenic seeds (positive for transgene expression) and controls to demonstrate RNAi-conferred resistance to fungal mycotoxin synthesis.

▶ 07:20
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