小菜蛾Argonaute2和Argonaute3在RNA干擾途徑中的作用
發(fā)布時間：2022-12-04 19:09
背景:RNA干擾（RNAi）是雙鏈RNA（dsRNA）介導(dǎo)的轉(zhuǎn)錄后基因沉默機制,已成為熱門的遺傳學(xué)工具用于研究動物、植物以及節(jié)肢動物上的基因功能。該過程可在轉(zhuǎn)錄水平上的細胞核內(nèi)發(fā)生,亦可通過RNA引導(dǎo)的沉默復(fù)合體（RISC）的轉(zhuǎn)錄后水平上的細胞質(zhì)中發(fā)生。Ago蛋白是RISC組成的核心酶,起到切割靶標(biāo)mRNA的作用。然而,Ago蛋白在小菜蛾上的功能還未有研究。因此,研究小菜蛾Ago蛋白在RNA干擾通路上的作用是很有必要的,也有助于提高鱗翅目昆蟲RNAi的效率。方法:從福州敏感品系小菜蛾（Plutella xylostella）的三齡幼蟲提取總RNA,以總RNA為模板通過反轉(zhuǎn)錄PCR擴增小菜蛾Ago2和Ago3基因,克隆PxAgo2和PxAgo3并送公司測序。利用多序列比對和構(gòu)建系統(tǒng)發(fā)生樹分析小菜蛾P(guān)xAgo2和PxAgo3功能域。構(gòu)建PxAgo2和PxAgo3表達載體用于小菜蛾細胞的瞬時表達。合成PxAgo2、PxAgo3,、PxActin Burs-α和EGFP基因的dsRNA,并注射小菜蛾三齡幼蟲,或轉(zhuǎn)染小菜蛾細胞系,以抑制靶標(biāo)基因的表達。利用qRT-PCR檢測基因在mRNA水平的表...
【文章頁數(shù)】：121 頁
【學(xué)位級別】：博士
【文章目錄】：
摘要
Abstract
1 Review of literature and objectives
1.1 RNA interference (RNAi)
1.2 Classes of RNAi
1.2.1 Small RNAs
1.2.2 Core components of RNAi
1.3 Ago proteins
1.3.1 Ago1 protein
1.3.2 Ago2 protein
1.3.3 Ago3 protein
1.4 RNAi process in insects
1.4.1 RNAi process in Lepidopteran insects
1.4.2 RNAi in Plutella xylostella
1.5 Objectives
2 Molecular cloning and functional characterization of Argonaute2 in RNAinterference pathway of Plutella xylostella
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials
2.2.2 Methods
2.3 Results
2.3.1 Identificaiton and characterization of the deduced amino acid sequence ofPxAgo2
2.3.3 PxAgo2 mRNA and protein expression level in P. xylostella
2.3.4 dsRNA-mediated suppression of PxAgo2 in P. xylsotella larvae
2.3.5 PxAgo2 knockdown highly reduces the RNAi efficiency in P. xylostella cells
2.3.6 PxAgo2 overexpression enhanced the RNAi efficiency in P. xylostellacells
2.4 Discussion
3 Molecular characterization and function of Argonaute3 RNA interferencepathway of Plutella xylostella
3.1 Introduction
3.2 Materials and methods
3.2.1 Molecular cloning and expression vector construction of PxAgo3
3.2.2 Phylogenetic tree and sequence analysis
3.2.3 dsRNA synthesis and injection into P. xylostella larvae
3.3 Results
3.3.1 Identification and sequence characterization of PxAgo3
3.3.2 PxAgo3 expression in response to dsRNA in P. xylostella larvae
3.3.3 PxAgo3 suppression decreased the RNAi efficiency in P. xylostella cells
3.3.4 PxAgo3 overexpression increased the RNAi efficiency in P. xylostella cells
3.4 Discussion
4 Discussion, conclusion and future perspectives
4.1 Discussion and conclusion
4.2 Future perspectives
References
Acknowledgements
Appedix
Published Papers and other Academic Achievements duringthe Ph.D. Program
本文編號：3708828
【文章頁數(shù)】：121 頁
【學(xué)位級別】：博士
【文章目錄】：
摘要
Abstract
1 Review of literature and objectives
1.1 RNA interference (RNAi)
1.2 Classes of RNAi
1.2.1 Small RNAs
1.2.2 Core components of RNAi
1.3 Ago proteins
1.3.1 Ago1 protein
1.3.2 Ago2 protein
1.3.3 Ago3 protein
1.4 RNAi process in insects
1.4.1 RNAi process in Lepidopteran insects
1.4.2 RNAi in Plutella xylostella
1.5 Objectives
2 Molecular cloning and functional characterization of Argonaute2 in RNAinterference pathway of Plutella xylostella
2.1 Introduction
2.2 Materials and methods
2.2.1 Materials
2.2.2 Methods
2.3 Results
2.3.1 Identificaiton and characterization of the deduced amino acid sequence ofPxAgo2
2.3.3 PxAgo2 mRNA and protein expression level in P. xylostella
2.3.4 dsRNA-mediated suppression of PxAgo2 in P. xylsotella larvae
2.3.5 PxAgo2 knockdown highly reduces the RNAi efficiency in P. xylostella cells
2.3.6 PxAgo2 overexpression enhanced the RNAi efficiency in P. xylostellacells
2.4 Discussion
3 Molecular characterization and function of Argonaute3 RNA interferencepathway of Plutella xylostella
3.1 Introduction
3.2 Materials and methods
3.2.1 Molecular cloning and expression vector construction of PxAgo3
3.2.2 Phylogenetic tree and sequence analysis
3.2.3 dsRNA synthesis and injection into P. xylostella larvae
3.3 Results
3.3.1 Identification and sequence characterization of PxAgo3
3.3.2 PxAgo3 expression in response to dsRNA in P. xylostella larvae
3.3.3 PxAgo3 suppression decreased the RNAi efficiency in P. xylostella cells
3.3.4 PxAgo3 overexpression increased the RNAi efficiency in P. xylostella cells
3.4 Discussion
4 Discussion, conclusion and future perspectives
4.1 Discussion and conclusion
4.2 Future perspectives
References
Acknowledgements
Appedix
Published Papers and other Academic Achievements duringthe Ph.D. Program
本文編號：3708828
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