Application of RNA Interference Technology to Acroporid Juvenile Corals

Ikuko Yuyama,Michio Hidaka,Tomihiko Higuchi

doi:10.3389/fmars.2021.688876

Abstract

Numerous genes involved in calcification, algal endosymbiosis, and the stress response have been identified in corals by large-scale gene expression analysis, but functional analysis of those genes is lacking. There are few experimental examples of gene expression manipulation in corals, such as gene knockdown by RNA interference (RNAi). The purpose of this study is to establish an RNAi method for coral juveniles. As a first trial, the genes encoding green fluorescent protein (GFP, an endogenous fluorophore expressed by corals) and thioredoxin (TRX, a stress response gene) were selected for knockdown. Synthesized double-stranded RNAs (dsRNAs) corresponding to GFP and TRX were transformed into planula larvae by lipofection method to attempt RNAi. Real-time PCR analysis to verify knockdown showed that GFP and TRX expression levels tended to decrease with each dsRNA treatment (not significant). In addition, stress exposure experiments following RNAi treatment revealed that planulae with TRX knockdown exhibited increased mortality at elevated temperatures. In GFP-knockdown corals, decreased GFP fluorescence was observed. However, the effect of GFP-knockdown was confirmed only in the coral at the initial stages of larval metamorphosis into polyps, but not in planulae and 1 month-old budding polyps. This study showed that lipofection RNAi can be applied to coral planulae and polyps after settlement, and that this method provides a useful tool to modify expression of genes involved in stress tolerance and fluorescence emission of the corals.

Highlights

RNA interference (RNAi) is a sequence-specific gene silencing phenomenon in which doublestranded RNA causes a reduction of the complementary RNA (Fjose et al, 2001)
As the initial knockdown trial in corals, TRX and green fluorescent protein (GFP) were used for RNAi targets
From A. tenuis planula larvae, cDNA clones that comprised a full-length open reading frame of GFP and TRX, respectively, were isolated, and double-stranded RNAs (dsRNAs) were synthesized from these clones

Summary

Introduction

RNA interference (RNAi) is a sequence-specific gene silencing phenomenon in which doublestranded RNA (dsRNA) causes a reduction of the complementary RNA (Fjose et al, 2001). RNAi experiments in hydra have revealed the genes involved in cephalic and foot development, budding, and axis determination (Lohmann et al, 1999; Amimoto et al, 2006; Galliot et al, 2007; Jakob and Schierwater, 2007; Chera et al, 2009). These findings have helped clarify how body plans evolved in metazoan organisms. Establishing RNAi technology for corals will enable further analysis of coral-specific phenomena, while morpholino oligo injection and CRISPR/Cas9-mediated genome editing are an promising approach for gene knockdown in corals (Yasuoka et al, 2016; Cleves et al, 2018)

Objectives

Methods

Results