小菜蛾Argonaute2和Argonaute3在RNA干扰途径中的作用

发布时间：2022-12-04 19:09

　　背景:RNA干扰（RNAi）是双链RNA（dsRNA）介导的转录后基因沉默机制,已成为热门的遗传学工具用于研究动物、植物以及节肢动物上的基因功能。该过程可在转录水平上的细胞核内发生,亦可通过RNA引导的沉默复合体（RISC）的转录后水平上的细胞质中发生。Ago蛋白是RISC组成的核心酶,起到切割靶标mRNA的作用。然而,Ago蛋白在小菜蛾上的功能还未有研究。因此,研究小菜蛾Ago蛋白在RNA干扰通路上的作用是很有必要的,也有助于提高鳞翅目昆虫RNAi的效率。方法:从福州敏感品系小菜蛾（Plutella xylostella）的三龄幼虫提取总RNA,以总RNA为模板通过反转录PCR扩增小菜蛾Ago2和Ago3基因,克隆PxAgo2和PxAgo3并送公司测序。利用多序列比对和构建系统发生树分析小菜蛾PxAgo2和PxAgo3功能域。构建PxAgo2和PxAgo3表达载体用于小菜蛾细胞的瞬时表达。合成PxAgo2、PxAgo3,、PxActin Burs-α和EGFP基因的dsRNA,并注射小菜蛾三龄幼虫,或转染小菜蛾细胞系,以抑制靶标基因的表达。利用qRT-PCR检测基因在mRNA水平的表... 

【文章页数】：121 页

【学位级别】：博士

【文章目录】：
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



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