Use of gene silencing for soybean bugs

Impact of dsRNA translocation potential in soybean pods for gene silencing studies in insects

29.04.2020 | 20:59 (UTC -3)

Soybean cultivation has become the most important agricultural chain for the Brazilian economy (MAPA, 2017), with pests and diseases that are increasingly becoming resistant to the chemical control methods used due to their indiscriminate use (Oliveira et al., 2016).

Following the discovery of molecules that act by silencing specific genes, known as RNA interference (RNAi), several methods of use and application have been and are being developed with the aim of revolutionizing control methods for various organisms (Hannon, 2002; Boutros & Ahringer, 2008).

The RNA interfering (RNAi) mechanism is a process that occurs naturally in eukaryotic organisms. This is a process that causes the silencing of genes before their translation, through the degradation of mRNA (Obbard, et al., 2009). This process was first described in plants and named post-transcriptional gene silencing, or PTGS (Jorgensen, et al., 1996).

RNAi has great biotechnological potential due to its high specificity, and can thus contribute to the field of entomology (Gordon & Waterhouse, 2007), whether for the functional study of genes or even as an alternative tool for controlling insect pests in agriculture ( Price & Gatehouse, 2008). In general, RNAi studies in these insects are carried out using plants genetically modified to express double-stranded RNAs (dsRNAs) or using exogenous molecules introduced through microinjection (Fishilevich et al., 2016).

This work aims to evaluate the feasibility of using the RNAi mechanism to study genes in insects, via food, in soybean pods. This methodology, known as “Ectopic Application”, is based on the direct absorption of dsRNA into plant tissue, which is subsequently absorbed by the insect after feeding on the plants, resulting in gene silencing. This is a less expensive methodology when compared to those traditionally used, which has already been successfully used in studies on other organisms and some insects. In this work, the translocation capacity of dsRNA in soybean pods was evaluated. For this, dsRNA molecules without complementarity with soybean transcripts were used.

The experiment was conducted at the Embrapa Soja biotechnology laboratory, in Londrina, PR, during 2017. To evaluate the translocation capacity of the dsRNA solution in the pods, 5 2 mL tubes were prepared with a microdrop at the bottom of the tubes. containing 50 µL of [FC1] solution with a concentration of 10 ng of GFP dsRNA. Afterwards, 5 standardized BRS 1001 BTRR soybean pods with 3 seeds were collected. Before introducing the pod into the tube, part of the peduncle was cut to keep the live part in contact with the solution. Subsequently, the pod stalk was placed in contact with the microdrop in the [FC2] inside the tube and stored for 24 hours in BOD at 26°C with a 14-hour photoperiod, with the volume being completely absorbed in this period of time. Afterwards, the pods were removed from the tubes, washed with distilled water, divided into three parts corresponding to the seed segments, the seed valves separated and frozen in liquid nitrogen. They were later placed in 2 mL tubes for identification and stored in a freezer at -80°C. The valves were separated and identified as follows: VX 1.1, 1.2, 1.3; VX 2.1, 2.2 and 2.3; VX 3.1, 3.2 and 3.3; VX 4.1, 4.2 and 4.3 and VX 5.1, 5.2 and 5.3 referring to the repetition of the pod after the section.

Total RNA from each section of pods was extracted individually using the Trizol method (Invitrogen®), followed by qualitative PCR reactions using specific primers for a 206bp fragment, to evaluate the presence of dsRNA in plant tissue. For each reaction, 100 ng of total RNA was used.

Through PCR analysis, the presence of dsRNA and its stability and integrity after 24h were observed (Figure 1), being mainly detected in sections of the pod close to the peduncle, where the dsRNA was administered. The stability of dsRNA 24 hours after application in plant tissue highlights the potential application of this technology both for gene validation assays in soybean insect pests and/or its potential use as a control tool.

Given the variation in the translocation profile between the 5 pods evaluated, it is suggested that more tests be conducted, both varying the dose and incubation time. Additionally, assays with maintenance of water flow, which may favor dsRNA translocation, can be considered.

Figure 1. Results of PCR and Electrophoresis reaction in sectioned soybean pods with GFP dsRNA translocation. VX 1.1 pod 1 section 1, VX 1.2 pod 1 section 2, VX 1.3 pod 1 section 3, VX 2.1 pod 2 section 1, VX 2.2 pod 2 section 2, VX 2.3 pod 2 section 3, VX 3.1 pod 3 section 1, VX 3.2 pod 3 section 2, VX 3.3 pod 3 section 3, VX 4.1 pod 4 section 1, VX 4.2 pod 4 section 2, VX 4.3 pod 4 section 3, VX 5.1 pod 5 section 1, VX 5.2 pod 5 section 2, VX 5.3 pod 5 section 3.
Figure 1. Results of PCR and Electrophoresis reaction in sectioned soybean pods with GFP dsRNA translocation. VX 1.1 pod 1 section 1, VX 1.2 pod 1 section 2, VX 1.3 pod 1 section 3, VX 2.1 pod 2 section 1, VX 2.2 pod 2 section 2, VX 2.3 pod 2 section 3, VX 3.1 pod 3 section 1, VX 3.2 pod 3 section 2, VX 3.3 pod 3 section 3, VX 4.1 pod 4 section 1, VX 4.2 pod 4 section 2, VX 4.3 pod 4 section 3, VX 5.1 pod 5 section 1, VX 5.2 pod 5 section 2, VX 5.3 pod 5 section 3.


Joan Brigo Fernandes, Agricultural Engineer, PhD student in Agronomy Plant Health, UEL

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