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ARS Home » Midwest Area » West Lafayette, Indiana » Crop Production and Pest Control Research » Research » Research Project #440652

Research Project: Genetic Analysis of a Candidate Hessian Fly-responsive Defense Gene Function Using a Rice Cultivar as Surrogate Genome

Location: Crop Production and Pest Control Research

Project Number: 5020-22000-019-004-S
Project Type: Non-Assistance Cooperative Agreement

Start Date: Sep 1, 2021
End Date: Aug 31, 2023

Objective:
Develop transgenic rice lines and evaluate phenotypic and molecular responses of transgenic plants and mutants to Hessian fly infestation.

Approach:
The coding sequence of the candidate Hessian fly-responsive defense gene from rice will be isolated from cDNA by PCR, while the upstream (~1kb) promoter sequence will be isolated from genomic DNA. Both sequences will be cloned into an appropriate cloning vector and sequence verified. To silence the gene CRISPR/Cas9 editing technology will be used. Calli generated from rice Japonica Kitaake cultivar will be transformed with the CRISPR constructs using Agrobacterium-mediated transformation with a goal to achieve ~70% mutation rate in the target gene. PCR amplification and sequencing will be undertaken in T0 lines to confirm mutagenesis in the target gene. Homozygous positive lines will be identified in T1 and T2 generations. At least two allelic mutants in the T3 generation lacking the Cas9 transgene will be selected for phenotypic analysis. Simultaneously, transgenic rice plants expressing a yellow fluorescence protein (YFP)-tagged gene fusion protein under the control of its native promoter will be generated for quantifying endogenous protein by introducing the constructs via Agrobacterium-mediated transformation. For this work, calli from both wild type Kitaake and CRISPR/Cas9 Kitaake mutants will be used. Phenotypic evaluation of transgenic lines and mutants will be done by infesting plants with Hessian fly Biotype L. Molecular characterization of these lines will be undertaken by gene expression analysis using quantitative real-time PCR.