Molecular characterization, expression analysis and RNAi knock-down of elongation factor 1α and 1γ fromNilaparvata lugens and its yeast-like symbiont

Abstract

In the present paper, four cDNAs encoding the alpha and gamma subunits of elongation factor 1 (EF-1) were cloned and sequenced from Nilaparvata lugens, named NlEF-1α, NlEF-1γ, and its yeast-like symbiont (YLS), named YsEF-1α and YsEF-1γ, respectively. Comparisons with sequences from other species indicated a greater conservation for EF-1α than for EF-1γ. NlEF-1α has two identical copies. The deduced amino acid sequence homology of NlEF-1α and NlEF-1γ is 96 and 64%, respectively, compared with Homalodisca vitripennis and Locusta migratoria. The deduced amino acid sequence homology of YsEF-1α and YsEF-1γ is 96 and 74%, respectively, compared with Metarhizium anisopliae and Ophiocordyceps sinensis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis revealed that the expression level of NlEF-1α and NlEF-1γ mRNA in hemolymph, ovary, fat body and salivary glands were higher than the midgut and leg tissue. YsEF-1α and YsEF-1γ was highly expressed in fat body. The expression level of NlEF-1α was higher than that of NlEF-1γ. Through RNA interference (RNAi) of the two genes, the mortality of nymph reached 92.2% at the 11th day after treatment and the ovarian development was severely hindered. The RT-qPCR analysis verified the correlation between mortality, sterility and the down-regulation of the target genes. The expression and synthesis of vitellogenin (Vg) protein in insects injected with NlEF-1α and NlEF-1γ double-stranded RNA (dsRNA) was significantly lower than control groups. Attempts to knockdown the YsEF-1 genes in the YLS was unsuccessful. However, the phenotype of N. lugens injected with YsEF-1α dsRNA was the same as that injected with NlEF-1α dsRNA, possibly due to the high similarity (up to 71.9%) in the nucleotide sequences between NlEF-1α and YsEF-1α. We demonstrated that partial silencing of NlEF-1α and NlEF-1γ genes caused lethal and sterility effect on N. lugens. NlEF-1γ shares low identity with that of other insects and therefore it could be a potential target for RNAi-based pest management.

Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data

“…The main function of EF-1γ is to ensure the correct scaffolding of different subunits in the EF-1 complex as well as to direct its intracellular localization [43]. The expression stability of EF-1γ had previously been evaluated in P. ginseng [34] and Nilaparvata lugens [44]. In our study, EF-1γ was one of the two most suitable reference genes with stable expression levels under various experimental conditions.…”

RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

“…IDGFs were confirmed to be the proteins cooperating with insulin that promote cell lineages derived from imaginal discs in Drosophila melanogaster (Kawamura et al, 1999;Varela et al, 2002;Zurovcová and Ayala, 2002). RNAi has been widely used to find out the functions of vital genes in different insects of economic importance (Tomoyasu and Denell, 2004;Chen et al, 2008;Gong et al, 2012;Asokan et al, 2013;Zhang et al, 2013;Qi et al, 2015;Wang et al, 2017;Ullah et al, 2020a). Recently, a study reported that silencing of IDGF6 in Bactrocera correcta through RNAi significantly decreases the expression of IDGF6, causes larval mortality and wing malformation in adult flies (Zhao et al, 2020).…”

Regulatory functions of trehalose-6-phosphate synthase in the chitin biosynthesis pathway in Tribolium castaneum (Coleoptera: Tenebrionidae) revealed by RNA interference

“…RNA interference (RNAi) is widely used to study gene function by suppressing gene expression via the injection of dsRNA (Fire et al , 1998). RNAi technology also has been widely used to investigate the functions of essential genes in insects (Tomoyasu & Denell, 2004; Chen et al ., 2008; Gong et al ., 2012; Wang et al ., 2017; Zhang et al ., 2013; Asokan et al ., 2013; Qi et al ., 2015), and has been particularly employed to silence vital genes (Belles, 2010; Liu et al , 2010; Wang et al , 2012). Similar studies using RNAi have resulted in diverse malformed phenotypes in insects and show varying efficiencies in the insect tissues investigated (Zhu et al , 2008; Belles, 2010; Scott et al , 2013; Xi et al , 2015).…”