两种寄主植物对斜纹夜蛾繁殖及卵黄原蛋白基因（Vg）表达的影响

发布时间：2018-08-30 07:50

【摘要】：斜纹夜蛾(Spodoptera litura Fabricius)是一种多食性害虫,全球范围内均有分布。近年来,斜纹夜蛾暴发增加危害严重。斜纹夜蛾的暴发性源于其具有较强的生殖力,单雌产卵量约在千粒,导致在极短的时间里暴发成灾。而寄主的多样性也为斜纹夜蛾提供了丰富的食料,为其大暴发奠定了基础。目前有很多研究探索不同寄主植物对斜纹夜蛾生殖的影响,但大多数都是从产卵量等生理生化指标上做出说明,很少挖掘与生殖相关的基因。而卵黄原蛋白基因作为一个与卵子发生密切相关的基因,在斜纹夜蛾中研究极少,也没有明确结果阐述不同寄主植物对Vg基因表达的影响及影响机制。本文就不同寄主植物对斜纹夜蛾生殖以及与生殖密切相关的Vg基因表达的影响入手,选取甘蓝和空心莲子草两种斜纹夜蛾喜食程度差异较大的寄主,主要研究其对斜纹夜蛾生殖相关指标的影响；采用荧光定量PCR技术、RT-PCR技术和SDS-PAGE技术,测定取食不同寄主斜纹夜蛾体内的Vg基因的表达、血淋巴中的卵黄原蛋白的含量；对取食不同寄主的斜纹夜蛾进行转录组分析,找出与Vg基因以及生殖调控相关联的关键基因,主要结果如下：1.取食不同寄主植物后,斜纹夜蛾的生殖指标均受到不同程度的影响。产卵前期表现为甘蓝空心莲子草,产卵历期、产卵量、潜在产卵量、雌虫寿命均保持一致趋势,为甘蓝空心莲子草。取食甘蓝后,产卵量约为取食空心莲子草斜纹夜蛾的2.7倍。而腹内残余卵量、产出率和孵化率差异不明显。成虫各阶段卵巢鲜重、生殖腺指数、卵巢管长度均为甘蓝空心莲子草。测定脂肪体和卵巢中的可溶性糖与蛋白,结果显示各个龄期的指标均为甘蓝空心莲子草。对两种寄主植株的主要营养成分,可溶性糖和蛋白测定得到,两者糖含量差异显著,甘蓝明显高于空心莲子草,而蛋白含量无显著差异。2.取食不同寄主植物后,斜纹夜蛾Vg基因的表达受到了不同程度的影响。Vg基因表现为雌虫脂肪体特异性表达。Vg表达的空间格局方面,在取食甘蓝的斜纹夜蛾中,Vg在第6天的蛹中开始转录,在第7天的蛹血淋巴中检测到蛋白条带。取食空心莲子草的斜纹夜蛾,Vg在第7天的蛹中开始转录,在第8天的蛹血淋巴中检测到蛋白条带。两者在Vg的表达量上,各个虫龄均存在显著差异。相差最大的阶段为24h成虫,取食甘蓝的斜纹夜蛾Vg mRNA相对表达量为4.33,取食空心莲子草的斜纹夜蛾Vg mRNA相对表达量为3.16,前者约为后者的1.37倍。3.对取食不同寄主植物的斜纹夜蛾进行转录组学分析,一共检测拼接出30671个基因序列,其中有15809个Unigene在数据库中有注释。对拼接的Unigene进行功能分类,在COG中以信号传导机制为主,在GO中以分子功能为主。KEGG分析发现Unigene主要映射于新陈代谢通路,说明寄主主要是通过影响斜纹夜蛾的新陈代谢过程从而影响昆虫的生长发育和繁殖。对羽化24h雌成虫进行对比,筛选出120个显著差异的基因,并通过Pathway分析发现存在11个具体的差异基因,选取了几个位于主路上的可能与生殖相关的基因,分别是：钙调蛋白(calmodulin)基因、尼曼匹克C2蛋白(Niemann-Pick C2 protein)基因和细胞色素P450 (cytochrome P450)基因,并进行了讨论。
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433.4

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