臺灣博碩士論文加值系統

秋行軍蟲(Spodoptera frugiperda)為寄主作物廣泛的農業害蟲，在台灣嚴重危害玉米和高粱等經濟作物的生產。目前針對秋行軍蟲的防治策略主要依賴噴灑化學藥劑。隨著食品安全以及環境保育等議題逐漸受到重視，開發環境有善的害蟲防治方法為現今重要的研究方向。本研究透過噴灑誘導基因靜默(spray-induced gene silencing, SIGS)的應用，以雙股RNA (dsRNA)誘發RNA干擾(RNA interference, RNAi)，靜默秋行軍蟲重要生理機能相關基因，並評估與蟲生真菌的協同防治成效。dsRNA進入昆蟲後的穩定性為影響昆蟲RNAi效率的因素之一，經測試秋行軍蟲中腸液具有降解dsRNA的能力，其中double-stranded ribonuclease 3 (dsRNase3)以及RNAi efficiency–related nuclease 3 (REase3)為中腸內表現量最高的核酸酶基因。本研究選擇秋行軍蟲Chitinase, Inhibitor of Apoptosis Protein (IAP)與Sucrose Non-Fermenting 7 (Snf7)為目標，以大腸桿菌(Escherichia coli) dsRNA生產系統進行目標基因片段dsRNA生產。透過餵食的方式對秋行軍蟲幼蟲施用dsRNA，與餵食滅菌水的對照組相比，在REase3, Chitinase, IAP以及Snf7相對基因表達降低了49 %至80 %，但對幼蟲的正常生長發育及存活未造成明顯影響。為了進一步提升RNAi的效果，本研究使用PEGylated-chitosan作為載體包覆dsRNA，並以相同的餵食方式對秋行軍蟲幼蟲進行測試，Chitinase以及IAP基因表達量分別下降了55 %與53 %，但在表現型上亦無顯著差異。隨後，研究測試了蟲生真菌Beauveria bassiana-NCHU-157與RNAi的協同防治效果，結果顯示無論是餵食還是噴灑dsRNA的RNAi策略，對蟲生真菌的殺蟲效果均無顯著提升。綜上所述，本研究中秋行軍蟲RNAi的效果尚不明確，雖然目標基因受到調降，但幼蟲的生長發育及與蟲生真菌的協同作用均未見明顯影響。對於秋行軍蟲體內核酸酶以及核心RNAi機制的深入研究將有助於未來RNAi技術在其防治中的應用。

Fall armyworm (Spodoptera frugiperda) is an agricultural pest with a wide host range, severely damaging the production of economically important crops such as corn, sorghum, and others in Taiwan. Current control strategies mainly rely on chemical pesticides. As food safety and environmental conservation issues become increasingly important, developing environmentally friendly pest control methods has become a critical research topic. This study plans to apply spray-induced gene silencing (SIGS) using double-stranded RNA (dsRNA) to induce RNA interference (RNAi), silencing key physiological genes in the fall armyworm. Additionally, it evaluates the synergistic control effectiveness with entomopathogenic fungi. The stability of dsRNA within insects is one factor affecting RNAi efficiency. Results showed that the midgut fluid of fall armyworms has the ability to degrade dsRNA. Among the ribonucleases, double-stranded ribonuclease 3 (dsRNase3) and RNAi efficiency-related nuclease 3 (REase3) are the most highly expressed in the fall armyworm midgut. In this study, we selected Chitinase, Inhibitor of Apoptosis Protein (IAP), and Sucrose Non-Fermenting 7 (Snf7) genes as targets to design dsRNAs. The production of dsRNA for these target gene fragments was using Escherichia coli dsRNA production system. In the RNAi assay, dsRNA was administered to fall armyworm larvae via feeding. The results demonstrated that the relative gene expression of REase3, Chitinase, IAP, and Snf7 decreased by 49% to 80% compared to the control treatment, respectively, where larvae were fed sterile water. However, no significant effects on larval growth, development, or survival rate were observed. To enhance the efficacy of RNAi, this study utilized PEGylated-chitosan as a carrier to encapsulate dsRNA, which was administered to fall armyworm larvae via the same feeding method. The results indicated a decrease in Chitinase and IAP gene expression by 55 % and 53 %, respectively. However, no significant phenotypic differences were observed. Subsequently, the study examined the synergistic control effect of the entomopathogenic fungus Beauveria bassiana-NCHU-157 with dsRNA. The results indicated that neither feeding nor spraying dsRNA significantly augmented the insecticidal efficacy of Beauveria bassiana. In conclusion, the RNAi efficacy on fall armyworm in this study remains inconclusive. While target gene expression was reduced, no significant impacts on larval growth, development, or synergistic interactions with Beauveria bassiana were observed. Further investigation into the ribonucleases and core RNAi mechanisms within the fall armyworm could aid the future application of RNAi technology in its control.

摘要　i
Abstract　ii
Contents　iv
List of tables　vi
List of figures　vii
1　Introduction　1
1.1　Fall armyworm Spodoptera frugiperda (J.E. Smith).　1
1.2　Control of fall armyworm.　1
1.3　RNA interference (RNAi).　2
1.4　RNAi application in pest control.　3
1.5　Challenges in applying RNAi for Lepidoptera pest control.　4
1.6　Ribonucleases in Lepidoptera.　5
1.7　Enhancing dsRNA delivery for RNAi applications.　6
2　Material and methods　8
2.1　Fall armyworm　8
2.2　Entomopathogenic fungi　8
2.3　Pathogenicity test of entomopathogenic fungi　9
2.4　Target gene selection for fall armyworm RNAi　9
2.5　Construction of dsRNA expression plasmid　10
2.6　dsRNA production, extraction, and purification.　11
2.7　Stability test of dsRNA　13
2.8　Evaluate ribonuclease genes in fall armyworm midgut　13
2.9　Synthesis of PEGylated-chitosan-dsRNA complex　14
2.10　RNAi assay with feeding method　15
2.11　Synergistic effect test of RNAi and Entomopathogenic fungi (EPF)　15
2.12　RNA extraction and RT-qPCR assay　16
2.13　Statistical analysis　17
3　Result　18
3.1　Screening of entomopathogenic fungi (EPF) against fall armyworm　18
3.2　Construct dsRNA expression plasmid and dsRNA production via E. coli.　18
3.3　DsRNA stability in the midgut of fall armyworm.　19
3.4　Primary ribonuclease genes expressed in fall armyworm midgut.　20
3.5　Fall armyworm RNAi with feeding strategy.　21
3.6　RNAi cooperation with entomopathogenic fungi.　22
3.7　PEGylated-chitosan as a carrier for enhancing the stability of dsRNA.　23
3.8　PEGylated-chitosan-dsRNA RNAi assay on fall armyworm.　24
3.9　PEGylated-chitosan-dsRNA cooperation with entomopathogenic fungi.　25
4　Discussion　27
5　Reference　32
6　Tables and figures　38

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