摘 要为研究胰岛素受体（Insulin Receptor，InR）基因调控褐飞虱海藻糖代谢平衡的机理，本文选取了褐飞虱的两个胰岛素受体基因InR1和InR2，采用RNA干扰技术分别或者同时抑制两基因的表达，在处理后48 h和72 h测定褐飞虱体内海藻糖、葡萄糖含量及海藻糖酶（Trehalase，简称TRE）活性的变化，同时检测海藻糖代谢途径中的一些关键基因的表达。结果显示，dsRNA注射后能够明显抑制InR本基因的表达，并导致褐飞虱蜕皮和翅膀发育困难；InR1低表达后72 h能够显著抑制两种糖类物质的含量，任意InR表达抑制后72 h，褐飞虱体内可溶性海藻糖酶（TRE1）活性上升，而膜结合型海藻糖酶（TRE2）活性下降；并且，当InR基因表达抑制后，检测到3个海藻糖酶基因和2个海藻糖合成酶基因（Trehalose-6-phophate synthase，简称TPS）的表达都表现出明显下降。这些结果说明胰岛素受体InR能够影响海藻糖等糖类物质的平衡。相关研究结果有助于更加全面的探索研究褐飞虱等昆虫胰岛素调控海藻糖及糖类物质平衡的潜在分子机理，为将来通过调控昆虫血糖平衡来控制害虫提供理论依据。
Abstract：In order to study the effect of insulin signaling pathway on the balance and development of trehalose metabolism in Nilaparvata lugens. The RNAi technique was used to inhibit the insulin receptor (InR) genes and its expressions were detected at 48 h and 72 h after injection. The changes of the content of trehalose, glucogen and trehalase activity were measured at 48 h and 72 h, as well as the expression level of some key genes, such as trehalose metabolic pathway genes were detected. The results showed that dsRNA could inhibit the expression of InR genes significantly, which caused the molt and wings development of the N. lugens. The low level expression of InR1 could significantly inhibit the contents of two carbohydrates of sugars. The activity of soluble trehalase (TRE1) increased at 72 h after inhibition of InR expression, while the membrane-bound trehalase activity (TRE2) decreased. At the same time, the expression of trehalase (TRE) and two trehalose-6-phophate synthase genes (TPS) were significantly decreased after inhibition of InR expression. These results suggest that InR can affect the balance of carbohydrate, such as trehalose. The results of this study can be helpful to study the potential molecular mechanism of the regulation of trehalose and carbohydrate balance in insects, such as N. lugens and other pest. And it can provide a theoretical basis for controlling pests by regulating the blood sugar balance of insects.
关键词：褐飞虱；胰岛素受体；RNAi；海藻糖代谢；
Keyword: Nilaparvata lugens; Insulin receptor; RNA interference; trehalose metabolism
目 录
摘要2
0引言4
1材料与方法5
1.1仪器与试剂5
1.1.1 主要仪器与设备5
1.1.2主要试剂与药品5
1.2褐飞虱虫源处理及水稻种植5
1.3实验方法5
1.3.1双链 RNA（dsRNA）的合成5
1.3.2褐飞虱显微注射6
1.3.3 dsRNA注射后褐飞虱的饲养6
1.4 各处理组中畸形褐飞虱虫体拍照及相关检测取材7
1.5 有关糖类物质参数测定7
1.5.1葡萄糖含量测定7
1.5.2海藻糖含量测定7
1.53海藻糖酶活测定7
1.6 胰岛素通路中相关基因表达情况的检测8
1.6.1 褐飞虱总RNA抽提8
1.6.2 cDNA 第一链的合成8
1.6.3 实时荧光定量 PCR8
1.7 数据分析9
2结果分析9
2.1 基因干扰的效果评价9
2.2 基因干扰后对两种糖类物质含量的影响9
2.3 基因干扰后对褐飞虱海藻糖代谢的影响10
3讨论11 胰岛素受体基因InR调控褐飞虱海藻糖代谢研究:http://www.chuibin.com/shengwu/lunwen_205576.html