RNAi (RNA interference, RNA 간섭)은 타겟 유전자의 mRNA 시퀀스와 상동한 이중 가닥 RNA (double-stranded RNA)에 의해 일어난다는 사실이 밝혀진 이후, 유전자의 기능을 파악하는 연구 도구로 활용되다가, 2018년에 비로소 사람의 유전자 치료제로 상용화가 되었다. 지금은 더 많은 질병들의 유전자 치료제를 만들기 위해 다양한 회사들이 임상을 진행하고 있다. 농업의 분야에서는 곤충의 ‘생존 필수 유전자’를 타겟함으로써 해충 방제제로 개발되어 서부 옥수수 뿌리벌레(Western corn rootworm)를 RNAi로 방제하기 위한 제품이 종자 형식으로 상용화 되었고, 최근에는 심각한 농약저항성을 가진 콜로라도 감자벌레(Colorado potato beetle)를 RNAi로 방제하기 위 해 ‘스프레이 방식의 뿌리는 dsRNA’가 상용화되었다. 추후에 더 많은 해충을 타겟으로 하는 제품이 개발될 예정 이다. 본 발표에서는 위에 언급한 상용화된 dsRNA 시퀀스의 환경안전성평가와 관련된 연구, off-target effect, siRNA 활용, nanoparticle 등 RNAi 기술을 보다 안전하고 유용하게 농업적으로 사용할 수 있는 방법에 대한 ‘실험 실의 역할’이 무엇인지 알아보고자 한다.

In moth, pheromone biosynthesis activating neuropeptide (PBAN) regulates pheromone biosynthesis by binding to its receptor (PBANr). In this study, we cloned a PBANr gene (Mvi-PBANr) from sex pheromone gland in M. vitrata, which encodes 475 amino acid and includes 7 transmembrane domains. As a results of phylogenetic analysis, Mvi-PBANr is clustered with lepidopteran PBANrs. Mvi-PBANr was investigated for the effect of pheromone biosynthesis via RNA interference (RNAi), gas chromatography (GC) and bioassay. Consequently, expression level of Mvi-PBANr suppressed via RNAi, resulting in decrease pheromone component (E10E12-16:Ald). Mating rate was also reduced when performing the RNAi. These results revealed that Mvi-PBANr played important role in the pheromone biosynthesis in M. vitrata, and Mvi-PBANr can be used as new pest control targets.

Prostaglandin E2(PGE2) is an autocrine and paracrine signal in insects and other animals. Its signal pathways in target cells are well understood in mammalian system but not in insects. Here, we assessed PGE2 signaling in hemocytes of Spodoptera exigua through knocking-down of signal component genes by RNA interference (RNAi) and knocking-out (KO) of PGE2 receptor using CRISPR-Cas9. From S. exigua transcriptome, we selected hemocyte signaling components and analyzed their functions in cellular immune responses through RNAi. KO mutant against PGE2 receptor exhibited severely hampered larval development and adult fecundity.

Frankliniella occidentalis is a major pest in agriculture. Following overuse of insecticides, high resistance has developed due to its high reproduction rate and short generation time. To control the resistant strains of the thrips, the ingestion RNAi- based control was established. A total of 67 genes were selected, and their double-stranded RNAs (dsRNA) were delivered to thrips via the leaf disc-feeding method. Among the genes screened, the dsRNA of Toll-like receptor 6 (TLR6) and coatomer protein subunit epsilon (COPE) resulted in the highest mortality (3.8- and 2.8-fold faster LT50 compared to control, respectively) when ingested by thrips. The dsRNA-fed thrips showed 53% and 83% reduced transcription levels of TLR6 and COPE, respectively. This result demonstrates that the observed mortality of thrips following dsRNA ingestion was due to RNAi, and this lethal genes can be employed as a practical tool to control thrips in the field.

Nitric oxide (NO) is an immune mediator in several insects. In addition, eicosanoids mediate various immune responses in response to microbial challenges. This study focuses on cross-talk between two immune mediators. Nitric oxide synthase (SeNOS) of Spodoptera exigua was predicted in its cDNA sequence by interrogation to the transcriptomes and validated by RT-PCR against developmental stage and larval tissues. RNA interference (RNAi) of SeNOS suppressed nodule formation of S. exigua larvae against heat-killed Xenorhabdus hominickii. RNAi of SeNOS also suppressed NO levels in fat body and hemocytes along with suppression of PLA2 activity. NOS-specific inhibitor, L-NAME, also inhibited PLA2 activity, but its enantiomer, D-NAME, did not. However, PLA2 of dexamethasone or oxindole did not suppress NO levels in the immune tissues. In addition, X hominickii did not inhibit up-regulation of NO levels. These results suggest that NO signal activates PLA2 to produce eicosanoids to mediate immune responses

복숭아순나방(Grapholita molesta)은 두 가지 주요 성페로몬 성분(Z-8-dodecenyl acetate and E-8-dodecenyl acetate)을 갖고 있다. 이 성 페로몬 성분의 생합성 과정 분석은 포화지방산의 10번 탄소에 이중결합을 합성하는 불포화효소(Δ10 DES)가 종 특이적 광학이성체 형성에 필수 적이라고 제시하였다. 그러나 이 효소의 분자적 특징에 대해서 분석되지 않았다. 본 연구는 복숭아순나방 성페로몬 샘의 전사체에서 Δ10 DES로 추정된 불포화효소(Gm-comp1575)의 단백질 기능 영역을 분석하였다. Gm-comp1575 유전자는 370개의 아미노산 서열 정보를 암호하고 있으며 분자량은 약 43.2 kDa 그리고 등전위점(pI)은 8.77로 추정되었다. 이 불포화효소는 4개의 막투과영역을 지니고 있으며, 6개의 탄수화물 결합 위 치가 아미노 말단과 세포내 영역에서 갖는 것으로 추정되었다. 분자계통분석은 Gm-comp1575가 다른 종에서 알려진 Δ10 DES와 유사성이 높은 것으로 밝혀졌다. Gm-comp1575 전사체는 암컷 성페로몬 샘 및 다른 복부 조직에서 발현되었다. 이 유전자 발현에 대한 RNA 간섭 처리는 처녀 암컷으로 하여금 사과원에서 수컷을 유인하는 능력을 크게 감소시켰다. 이러한 결과는 Gm-comp1575가 복숭아순나방의 성페로몬 생합성과 관련 이 있는 유전자라고 제시하고 있다.

Beauveria bassiana JEF-007 with strong virulence against Riptortus pedestiris was selected for the Agrobacerium tumefaciens-mediated transformation(AtMT). AtMT generated two transformants, B1-06 and C1-49, showed significantly reduced virulence against R. pedestris. To identify the virulence-related genes, thermal asymmetric interlaced(TAIL) PCR and flanking region analysis were performed. From the analysis, two genes, Complex I intermediated-associated protein 30(CIA30) and Autophagy protein 22(Atg22), possibly related virulence in B. bassiana JEF-007. For the analysis of two putative virulence-related genes in JEF-007, hairpin RNA interference (hpRNAi) is under consideration. This work can provide the functional roles of the virulence-related genes in B. bassiana JEF-007.

Integrin is a heterodimer protein that locates on cell membrane to interact with neighboring cells or extracellular matrix. A transcriptome analysis of the brassica leaf beetle, Phaedon brassicae, midgut identified both α and β subunits of integrin. RNA interference of β subunit genes significantly impaired survival of both larvae and adults of P. brassicae. A recombinant bacteria expressing double-stranded RNA specific to β integrin of P. brassicae were constructed and showed significant oral toxicities.

Expression of hairpin RNA corresponding to the part of COPA transcript was done by agroinfiltration in soybean plants and was confirmed by qRT-PCR. In a pot experiment, T. urticae was infested on agroinfiltrated soybean plants and T. urticae mortality was observed and compared with control plants overtime. Significantly higher mortalities of T. urticae were observed in the COPA-agroinfiltrated soybean plants from post-infestation day 2 (15 ±5%), day 4 (50 ±10 %). At post-infestation day 6, mortality reached to (70 ± 15%). To validate the observed COPA silencing effect in T. urticae fed on the agroinfiltrated soybean plant expressing COPA hairpin RNAs, qRT-PCR analysis was performed. The transcript level of COPA gene was decreased in T. urticae fed on agroinfiltrated soybean plants expressing COPA hairpin RNA from post-infestation day 2. At post-infestation day 2, 4 and 6, COPA transcript levels were reduced by 23.8, 20.7 and 18.8 fold, respectively compared to post-infestation day 1 (control). The results obtained in this study also ruled that the plant mediated production and uptake of silencing (dsRNAs/siRNAs) is an effective way to trigger RNAi in the T. urticae.

Due to its rapid development of resistance to nearly all arrays of acaricide, Tetranychus urticae is extremely hard to control using conventional acaricides. As an alternative control measure of acaricide-resistant mites, RNA interference (RNAi)-based methods have recently been suggested. A double-stranded RNA (dsRNA) delivery method using multi-unit chambers was established and employed to screen the RNAi toxicity of 42 T. urticae genes. Among them, the dsRNA treatment of coatomer I (COPI) genes, such as coatomer subunit epsilon (COPE) and beta 2 (COPB2), resulted in high mortality [median lethal time (LT50) = 89.7 and 120.3h, respectively]. The transcript level of the COPE gene was significantly (F3,9 = 16.2, P =0.001) reduced by up to 24% following dsRNA treatment, suggesting that the toxicity was likely mediated by the RNAi of the target gene. As a toxicity enhancement strategy, the recombinant dsRNA was generated by reciprocally recombining half-divided fragments of COPE and COPB2. The two recombinant dsRNAs exhibited higher toxicity than the respective single dsRNA treatments as determined using LT50 values (79.2 and 81.5h, respectively). This finding indicates that the recombination of different genes can enhance RNAi toxicity and be utilized to generate synthetic dsRNA with improved RNAi efficacy.

Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice transmitted by small brown planthopper, SBPH, Laodalphax striatellus. RNA interference (RNAi) is an universal gene-knockdown mechanism in eukaryotic organisms which includes insects，and has been considered as an alternative strategy to control insect pests. Hence, we applied this technique to interfere the translation of target RNA genes to knockdown the virus gene on RSV-viruliferous L. striatellus. Three out of seven RSV genes, RdRp, NS3, and NCP were used as target genes and each dsRNA targeting the viral genes were delivered to the insects indirectly through the rice leaves by irrigation. As a result, not only the relative expression level of target genes decreased but also those of non-target genes and the replication of RSV genome as well. In summary, leaf-mediated dsRNA feeding methods would be useful in the knockdown of target genes on piercing-sucking insects. The genes used in this experiment can be utilized for the development of pest-resistant transgenic plants based on RNAi.

RNA interference (RNAi)는 double stranded RNA (dsRNA)를 이용하여 유전자 발현을 억제함으로써 특정 유전자를 침묵시키는 유전자 조절 기법이다. RNAi에 의한 유전자 발현억제는 생명체 내에 침입한 바이러스와 점핑유전자들이 가지는 위험으로부터 유전자 발현을 조절하여 게놈을 보호한다. 이러한 RNAi에 의한 유전자 발현억제는 크게 세 단계로 구성되어 있으며, (1) dsRNA가 Dicer라는 단백질에 의해 인식되어 siRNA (small interfering RNA)로 만들어지는 단계, (2) 이 siRNA들이 단백질 복합체인 RISC (RNA induced silencing complex)와 결합하는 단계, (3)이렇게 만들어진 복합체가 자연적으로 생성되는 mRNA에 결합하여 그 가닥들을 잘라내고 분해하는 단계를 거침으로써 모 유전자의 발현을 억제시킨다. 따라서 RNAi를 이용한 해충방제 기술은 근본적으로 적용가능한 치사유전자의 선발과 곤충체내로 효과적인 dsRNA 전달체계의 개발이 필요하다. 본 연구에서는 세계적으로 널리 분포하며 방제가 어려운 해충인 파밤나방 (Spodoptera exigua)을 대상으로 곤충의 세포성면역반응과 세포와 세포간 대화에 중요한 단백질인 integrin의 발현에 관여하는 integrin subunit β1 (Seintβ1) 유전자를 침묵시킴으로써 살충 효과를 확인할 수 있었다. 이를 토대로 효과적인 dsRNA 전달을 위한 유전자도입 작물의 개발을 목표로 대장균 (Escherichia coli HT115)을 이용한 Seintβ1에 특이적인 dsRNA의 in vivo 생산과 파밤나방 3령 유충을 대상으로 한 dsRNA의 섭식실험에서 integrin 유전자의 발현억제가 확인되었다.

Teratocytes (TCs) are the cells derived from the embryonic serosal membrane of some parasitic hymenopteran insects. As a parasitic factor, TCs are multifunctional in host regulation by inducing nutritional, immune, and developmental alterations. However, little is understood about their genetic constituents. This study reveals a comprehensive view of the genes expressed by TCs through a transcriptome analysis based on RNAseq technology. More than 6.29 Gb sequences were used to assemble 34,686 contigs (>200 bp) and annotated into different functional categories. The TC transcriptome profile was clearly distinct from those of hemocytes and the fat body. The TC transcriptome contained components of insulin signaling and biosynthesis of juvenile hormone and 20-hydroxyecdysone. TCs also expressed various groups of digestive enzymes, supporting its nutritional role for the growing parasitoid larvae in parasitism. Furthermore, this transcriptome analysis annotated two kinds of immunosuppressive serine protease inhibitors (serpins) and Rho GTPase-activating proteins (RhoGAPs). To determine the biological functions of these factors, we devised ex vivo RNA interference (RNAi) by conducting knockdown of gene expression in in vitro cultured TCs followed by injection of the treated TCs to test insects. Ex vivo RNAi revealed that some serpins and RhoGAPs expressed in TCs inhibited host cellular immunity. This study reports a transcriptome of the unique TC animal cell, and its immunosuppressive genetic factors using ex vivo RNAi technology.

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2,517 bp) of a integrin subunit β1 (HaITGβ1) was cloned from the oriental tobacco budworm, Helicoverpa assulta. Phylogenetic analysis showed that HaITGβ1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (61%) to β1 of other Noctuidae such as Spodoptera exigua and S. litura. Structural analysis of the HaITGβ1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that HaITGβ1 was expressed in all developmental stages and all tested tissues of H. assulta. Injection of double-stranded HaITGβ1 RNA (dsHaITGβ1) into third instar of H. assulta suppressed HaITGβ1 expression and resulted in significant delay from last larval stage to pupal stage. The dsHaITGβ1 injection significantly impaired nodule formation of H. assulta in response to bacterial challenge and hemocyte adherence. These results suggest that HaITGβ1 plays crucial roles in cellular immune responses as well as development in H. assulta.