【摘要】：斜紋夜蛾(Spodoptera litura Fabricius)是一種多食性害蟲,全球范圍內(nèi)均有分布。近年來,斜紋夜蛾暴發(fā)增加危害嚴重。斜紋夜蛾的暴發(fā)性源于其具有較強的生殖力,單雌產(chǎn)卵量約在千粒,導(dǎo)致在極短的時間里暴發(fā)成災(zāi)。而寄主的多樣性也為斜紋夜蛾提供了豐富的食料,為其大暴發(fā)奠定了基礎(chǔ)。目前有很多研究探索不同寄主植物對斜紋夜蛾生殖的影響,但大多數(shù)都是從產(chǎn)卵量等生理生化指標上做出說明,很少挖掘與生殖相關(guān)的基因。而卵黃原蛋白基因作為一個與卵子發(fā)生密切相關(guān)的基因,在斜紋夜蛾中研究極少,也沒有明確結(jié)果闡述不同寄主植物對Vg基因表達的影響及影響機制。本文就不同寄主植物對斜紋夜蛾生殖以及與生殖密切相關(guān)的Vg基因表達的影響入手,選取甘藍和空心蓮子草兩種斜紋夜蛾喜食程度差異較大的寄主,主要研究其對斜紋夜蛾生殖相關(guān)指標的影響；采用熒光定量PCR技術(shù)、RT-PCR技術(shù)和SDS-PAGE技術(shù),測定取食不同寄主斜紋夜蛾體內(nèi)的Vg基因的表達、血淋巴中的卵黃原蛋白的含量；對取食不同寄主的斜紋夜蛾進行轉(zhuǎn)錄組分析,找出與Vg基因以及生殖調(diào)控相關(guān)聯(lián)的關(guān)鍵基因,主要結(jié)果如下：1.取食不同寄主植物后,斜紋夜蛾的生殖指標均受到不同程度的影響。產(chǎn)卵前期表現(xiàn)為甘藍空心蓮子草,產(chǎn)卵歷期、產(chǎn)卵量、潛在產(chǎn)卵量、雌蟲壽命均保持一致趨勢,為甘藍空心蓮子草。取食甘藍后,產(chǎn)卵量約為取食空心蓮子草斜紋夜蛾的2.7倍。而腹內(nèi)殘余卵量、產(chǎn)出率和孵化率差異不明顯。成蟲各階段卵巢鮮重、生殖腺指數(shù)、卵巢管長度均為甘藍空心蓮子草。測定脂肪體和卵巢中的可溶性糖與蛋白,結(jié)果顯示各個齡期的指標均為甘藍空心蓮子草。對兩種寄主植株的主要營養(yǎng)成分,可溶性糖和蛋白測定得到,兩者糖含量差異顯著,甘藍明顯高于空心蓮子草,而蛋白含量無顯著差異。2.取食不同寄主植物后,斜紋夜蛾Vg基因的表達受到了不同程度的影響。Vg基因表現(xiàn)為雌蟲脂肪體特異性表達。Vg表達的空間格局方面,在取食甘藍的斜紋夜蛾中,Vg在第6天的蛹中開始轉(zhuǎn)錄,在第7天的蛹血淋巴中檢測到蛋白條帶。取食空心蓮子草的斜紋夜蛾,Vg在第7天的蛹中開始轉(zhuǎn)錄,在第8天的蛹血淋巴中檢測到蛋白條帶。兩者在Vg的表達量上,各個蟲齡均存在顯著差異。相差最大的階段為24h成蟲,取食甘藍的斜紋夜蛾Vg mRNA相對表達量為4.33,取食空心蓮子草的斜紋夜蛾Vg mRNA相對表達量為3.16,前者約為后者的1.37倍。3.對取食不同寄主植物的斜紋夜蛾進行轉(zhuǎn)錄組學(xué)分析,一共檢測拼接出30671個基因序列,其中有15809個Unigene在數(shù)據(jù)庫中有注釋。對拼接的Unigene進行功能分類,在COG中以信號傳導(dǎo)機制為主,在GO中以分子功能為主。KEGG分析發(fā)現(xiàn)Unigene主要映射于新陳代謝通路,說明寄主主要是通過影響斜紋夜蛾的新陳代謝過程從而影響昆蟲的生長發(fā)育和繁殖。對羽化24h雌成蟲進行對比,篩選出120個顯著差異的基因,并通過Pathway分析發(fā)現(xiàn)存在11個具體的差異基因,選取了幾個位于主路上的可能與生殖相關(guān)的基因,分別是：鈣調(diào)蛋白(calmodulin)基因、尼曼匹克C2蛋白(Niemann-Pick C2 protein)基因和細胞色素P450 (cytochrome P450)基因,并進行了討論。
[Abstract]:Spodoptera litura Fabricius (Spodoptera litura Fabricius) is a polyphagous pest distributed worldwide. In recent years, the outbreak of Spodoptera litura has increased severely. Many studies have been carried out to explore the effects of different host plants on the reproduction of Spodoptera litura, but most of them are based on physiological and biochemical indicators, such as oviposition, and rarely on genes related to reproduction. In this paper, the effects of different host plants on the reproduction of Spodoptera litura and the expression of Vg genes closely related to reproduction were studied, and the preference of two species of Spodoptera litura was selected. The effects of different host on reproductive indexes of Spodoptera litura were studied. The Vg gene expression and vitellogenin content in the haemolymph of Spodoptera litura fed on different hosts were detected by fluorescence quantitative PCR, RT-PCR and SDS-PAGE techniques. The main results were as follows: 1. After feeding on different host plants, the reproductive indexes of Spodoptera litura were affected to varying degrees. After feeding on cabbage, the oviposition was about 2.7 times as much as that of Spodoptera litura. However, there was no significant difference in the number of eggs remaining in the abdomen, fertility and hatching rate. The results showed that the main nutrient components, soluble sugar and protein of the two host plants were significantly different. The content of sugar in cabbage was significantly higher than that in lotus, but there was no significant difference in protein content. 2. Vg gene expression of Spodoptera litura was not detected after feeding on different host plants. In the spatial pattern of Vg expression, Vg began to transcribe in the 6th day pupa and protein bands were detected in the 7th day pupa haemolymph of Spodoptera litura feeding on cabbage. In Spodoptera litura, Vg began to transcribe in the 7th day pupa and in the 8th day pupa. Protein bands were detected in pupa haemolymph. There were significant differences in Vg expression among different instars. The largest difference was 24 h adults. The relative expression of Vg mRNA of Spodoptera litura fed on cabbage was 4.33. The relative expression of Vg mRNA of Spodoptera litura fed on water lily was 3.16, which was 1.37 times of that of Spodoptera litura fed on water lily. The transcriptome analysis of Spodoptera litura showed that 30671 gene sequences were mosaic, of which 1589 Unigenes were annotated in the database. The functional classification of the mosaic Unigenes was carried out. The signal transduction mechanism was dominant in COG, and the molecular function was dominant in GO. By comparing the female adults after emergence for 24 hours, 120 genes with significant differences were screened out. Pathway analysis showed that there were 11 specific genes, and several possible reproductive pathways were selected. The related genes are calmodulin gene, Niemann-Pick C2 protein gene and cytochrome P450 gene.
【學(xué)位授予單位】：揚州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S433.4

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