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摘要：马铃薯甲虫是一种世界性检疫对象和重大外来入侵物种，在我国主要分布在新疆，可危害多种茄科作物，是马铃薯上最重要的害虫。保幼激素(Juvenile Hormone,JH)是一类保持昆虫幼虫性状和促进成虫卵巢发育的激素。保幼激素由咽侧体分泌，和蜕皮激素(20-hydroxyecdysone,20E)协同调控昆虫发育、变态与生殖。马铃薯甲虫的发育、变态、繁殖等受保幼激素和蜕皮激素的调控，但是保幼激素和蜕皮激素的调控机理有待进一步研究。之前有研究表明Groucho可能参与到保幼激素和蜕皮激素的信号通路中。本论文主要研究Groucho在马铃薯甲虫的保幼激素和蜕皮激素通路中的作用，实验者通过克隆得到Groucho全长序列并进行进化分析，确定该基因的时空表达模式，最后检测了Groucho对保幼激素和蜕皮激素信号的表达响应。本文结果为该基因功能的进一步研究奠定了基础。30493
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关键词：马铃薯甲虫；保幼激素；蜕皮激素
Identification of Groucho and its transcriptional response to juvenile hormone and 20-hydroxyecdysone signaling in Leptinotarsa decemlineata larvae
Abstract：Leptinotarsa decemlineata (say) was a cosmopolitan quarantine and significant alien invasive insect species in China. The beetle was mainly distributed in Xinjiang, it fed on various solanaceous crops and has become the most important pest in potatoes. Juvenile hormone (JH) was secreted by corpus allatum, It was involved in the regulation of the development, reproduction and metamorphosis in Leptinotarsa decemlineata, together with 20-hydroxyecdysone (20E). However, up to now, the underlining mechanisms were not fully clear and deserved further researches. A previous research revealed that a gene named Groucho participated in JH signaling pathway. In the present paper, the full-length Groucho cDNA was cloned, its evolutionary relationship was evaluated. Moreover, the temporal and spatial expression patterns of this gene were examined. Finally, the transcriptional response of Groucho was tested to the signaling pathway of juvenile hormone and 20-hydroxyecdysone. These results will facilitate further researches on the function of Groucho.
Key words: Leptinotarsa decemlineata ; juvenile hormone ; 20-hydroxyecdysone
目 录
摘要 1
关键词 1
Abstract 1
Key words 1
引言 1
1 材料与方法 2
1.1供试昆虫 2
1.2 实验材料和仪器设备 2
1.2.1 实验材料 2
1.2.2 仪器设备 3
1.3 马铃薯甲虫中Groucho基因全长序列的克隆 4
1.3.1 PCR引物的设计 4
1.3.2 PCR扩增目的片段 4
1.3.3 PCR产物的回收纯化 4
1.3.4重组载体的构建 5
1.3.5重组载体的转化与菌液培养 5
1.3.6菌液PCR与测序 5
1.3.7 5’RACE 扩增引物设计 6
1.3.8 RACE获得目的基因全长 6
1.3.9 RACE产物的回收、连接、转化、菌液培养和测序 7
1.4 马铃薯甲虫Groucho基因的生物信息学分析 7
1.4.1 氨基酸多重序列比对 7
1.4.2 进化树的构建 7
1.5 Q-PCR确定Groucho基因的时空表达模式和对保幼激素和蜕皮激素的表达响 8
1.5.1 Q-PCR引物的设计 8
1.5.2 Q-PCR最适引物的验证 8
1.5.3 确定Groucho的时空表达模式 8
1.5.4 检测Groucho对保幼激素和蜕皮激素的表达响应 8
2 结果与分析 8