Location: Crop Genetics and Breeding Research
Project Number: 6048-21000-032-002-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Apr 1, 2023
End Date: Mar 31, 2028
Objective:
1. Clone four target genes into an E. coli expression vector for dsRNAs production.
2. Optimize the conditions for dsRNA application to achieve the most effectiveness using laboratory kernel/pods bioassay.
3. Determine the effectiveness of these dsRNA in reducing fungal infection and aflatoxin production under artificial inoculation conditions in greenhouse and samll-scale field studies.
4. Screen and characterize different types of nanoparticles and dsRNA system in preventing aflatoxin production.
Approach:
1. Clone the four target genes, as initial step, into an E. coli expression vector for dsRNAs production. A 300-500 bp fragment from each of these four genes will be selected and amplified by PCR using cDNA from A. flavus and primer sets containing Sac I restriction site at the 5’ end and Xho I at 3’ end. These PCR products will be digested and ligated to the corresponding sites in the L4440 vector. In addition, a peanut phytoene desaturase (PDS) gene will also be cloned in the same manner. The production of dsRNA by E. coli HT115 (DE3) cells for each target gene will be performed in 6 liters of LB medium.
2. Determine the effectiveness of dsRNA uptake into peanut leaves and pods/seeds delivered through different methods. The dsRNA targeting the peanut PDS gene will be used as a positive control to visually assess the efficiency of different dsRNA delivery methods: (a) different concentrations (100~500 µg/mL); (b) using an airbrush with different pressures (60 psi to 120 psi at 2~3 cm away from leaf surface); and (c) coating dsRNA with lignin-based nanoparticles that are being developed in Dr. Chen’s lab. This study will be conducted in growth chambers with eight 3-week-old peanut plants per treatment. If a consistent bleached phenotype is observed, the corresponding condition(s) will be used. Laboratory peanut kernels/pods bioassay will be carried by using surface-sterilized peanut kernels and pods coated with dsRNA at the concentrations described above. The dsRNA from bacterial cells containing an empty vector will be used as control. After kernels air-dried under the biosafety hood, corn kernels will be inoculated with freshly harvested A. flavus conidia suspension (5 x106/mL) and incubated for 7 days before being dried for aflatoxin analysis using HPLC. The fungal biomass will be determined using real time PCR with genomic DNA extracted from tested kernels as templates.
3. Examine the efficacy of these dsRNAs in suppressing A. flavus infection and aflatoxin contamination of peanut in the laboratory and under greenhouse/field conditions. The ability of some of these dsRNAs in suppressing A. flavus growth and/or aflatoxin production has been demonstrated in our previous studies involving transgenic corn plants expressing a construct to produce dsRNAs against these genes. For evaluating these dsRNAs against A. flavus under growth chamber conditions, eight plants will be sprayed with 1 mL/plant of dsRNA at four different concentrations (50, 100, 200, and 400 µg/mL) for each target gene. DsRNAs will be sprayed 24 hr prior to inoculating the peanut plants with spore suspension of A. flavus (5 x 106 spores/mL). Peanut leaves sprayed with dsRNA produced from the empty vector (EV) and inoculated will be included as controls. Aflatoxin production and fungal growth inside infected peanut kernels compared to the controls will be evaluated when the peanut plants are mature. If successful, the study will be conducted again under greenhouse conditions using the optimum concentration of dsRNA determined above. Small scale field studies will be initiated in year 2 and year 3.
