桔小实蝇胰岛素信号通路基因表达特性及功能研究

发布时间：2018-01-13 20:34

本文关键词：桔小实蝇胰岛素信号通路基因表达特性及功能研究　出处：《西南大学》2016年硕士论文　论文类型：学位论文

【摘要】：桔小实蝇Bactrocera dorsalis(Hendel)隶属双翅目Diptera、实蝇科Tephritidae,是一种广泛分布于热带和亚热带地区的世界性农业害虫。该虫寄主范围广、适应能力强、繁殖力高且扩散能力强,是国际贸易的技术壁垒,被许多国家列为检疫对象。桔小实蝇产卵于果实内,幼虫潜居于果瓤中取食危害,导致果实腐烂、未熟先落,降低果蔬的经济价值和食用价值。目前,由于化学药剂的不科学使用,桔小实蝇对多种常用杀虫剂产生严重的抗药性。因此,亟待寻找新的杀虫剂作用靶标或研发绿色防控技术。昆虫胰岛素信号的转导能够调节机体的生长发育、代谢、生殖以及衰老等重要生理过程。因此,阐明桔小实蝇胰岛素信号通路调控其变态发育的分子机制,可为寻找杀虫剂新的作用靶标提供理论基础。本学位论文以桔小实蝇为研究对象,利用RT-qPCR技术分析胰岛素信号通路7个重要基因在桔小实蝇各生长发育时期和体段或组织中的表达模式;分析这些基因在20E和饥饿胁迫后的应激表达模式;利用RNAi技术解析这些基因的功能;最后,利用HPLC技术检测沉默桔小实蝇BdFoxO后对其体内蜕皮激素滴度的影响,进一步阐明胰岛素信号通路基因与蜕皮激素间的关联。研究结果将明确这些基因的功能以及调控方式,为桔小实蝇的持续防控提供新的思路和方法。主要研究结果如下:1桔小实蝇胰岛素信号通路3个基因的时空表达模式分析1.1桔小实蝇胰岛素信号通路3个基因在各发育时期的表达模式利用RT-qPCR技术解析胰岛素信号通路3个重要基因(BdFoxO、BdFBP、BdPEPCK)在桔小实蝇的各发育时期的mRNA表达模式。结果表明,BdFoxO在幼虫及蛹期的相对表达量较高,且最高表达量出现在幼虫孵化后的第1天;BdFBP在幼虫阶段的相对表达量较高,其余发育时期间差异不显著;BdPEPCK在蛹期相对表达量较高,成虫羽化后第1天的表达量最高。结合前期研究结果发现大多数胰岛素信号通路基因在桔小实蝇幼虫及蛹期的相对表达量较高,说明这些基因参与调控桔小实蝇的蜕皮过程。1.2桔小实蝇胰岛素信号通路3个基因在不同体段或组织中的表达模式利用RT-qPCR技术解析胰岛素信号通路3个重要基因(BdFoxO、BdFBP、BdPEPCK)在桔小实蝇头部、胸部、腹部、卵巢、马氏管、脂肪体及中肠7个不同体段或组织中的mrna表达模式。结果表明,bdfoxo在中肠相对表达量最高,其后为脂肪体。bdfbp和bdpepck在脂肪体中表达量最高,且bdpepck在脂肪体的表达量是卵巢表达量的102.1倍。脂肪体能够整合多种营养和激素信号调控昆虫个体和细胞大小,胰岛素信号通路基因在桔小实蝇脂肪体相对较高水平的表达进一步说明这些基因可能影响其生长发育过程。220e和饥饿处理对桔小实蝇胰岛素信号通路7个基因表达的影响2.120e处理对桔小实蝇胰岛素信号通路7个基因表达的影响利用微量注射法,对桔小实蝇第5日龄幼虫注射不同剂量的20e,通过rt-qpcr技术检测胰岛素信号通路7个基因(akt、pdk、pi3k21b、inr、bdfoxo、bdfbp以及bdpepck)的相对表达量。结果表明,除akt在20e处理后相对表达水平显著下调外,其余6个基因总体呈显著上调趋势。说明在外源激素的刺激下,昆虫体内胰岛素信号通路基因转录水平出现相应调节变化,使体内各激素间达到动态平衡,从而维持自身体内的稳定。2.2饥饿处理对桔小实蝇胰岛素信号通路7个基因表达的影响利用rt-qpcr技术解析桔小实蝇胰岛素信号通路7个基因在饥饿处理后的表达水平变化。结果表明,除akt及bdfbp在饥饿处理后显著下调外,其余多数基因的表达显著上调。推测桔小实蝇幼虫在受到饥饿胁迫后,可能通过胰岛素信号通路基因间转录水平的相互协调加强体内信号转导,从而避免不良环境对自身种群延续产生的威胁。3桔小实蝇胰岛素信号通路7个基因的功能初探通过体外转录法获得高质量dsrna,借助微量注射法将外源dsrna导入桔小实蝇4日龄幼虫体内。rt-qpcr分析结果表明,7个基因48h后沉默效率均在50%左右,沉默效率最高的在80%以上。分别检测7个胰岛素信号通路基因的表达水平,发现7个基因相互之间存在不同程度的影响,说明这些基因之间可能存在一系列反馈与负反馈调节机制。沉默后对虫体进行表型观察,发现大多胰岛素信号通路基因沉默后对其幼虫化蛹时间产生一定的影响,推测与胰岛素信号通路的基因沉默导致桔小实蝇发生某些生理变化有关。通过对注射dsbdfoxo的幼虫进一步观察发现,沉默bdfoxo的幼虫体重明显变重,虫体增大。这一结果进一步说明胰岛素信号通路基因对桔小实蝇生长发育具有重要作用。4桔小实蝇BdFoxO基因对其体内蜕皮激素滴度的影响利用HPLC技术检测BdFoxO基因RNAi后桔小实蝇体内蜕皮激素滴度的变化情况。结果发现,桔小实蝇幼虫BdFoxO基因沉默后,其体内20E含量极显著降低,这可能是桔小实蝇BdFoxO基因沉默后导致化蛹时间延迟的原因。
[Abstract]:Bactrocera dorsalis (Hendel) Diptera belongs to Diptera, Tephritidae Tephritidae, is a widely distributed in tropical and subtropical regions of the world agricultural pests. The insect host range, strong adaptability, high fecundity and dispersal ability is strong, the technical barriers to international trade, many countries are classified as quarantine. Eggs in the fruit fly larvae, feeding fruit pulp in the latent harm, cause fruit rot and immature first fall, reducing the economic value of fruits and vegetables and edible value. At present, because the chemicals are not scientific, the Bactrocera dorsalis produced serious resistance to various insecticides. Therefore, it is urgent to find targets of pesticide or research and development of new green control techniques. The insulin signal transduction of insects to regulate the body's metabolism, growth, reproduction and aging and other important physiological processes. Therefore, to clarify its The molecular mechanism of regulation of insulin signaling pathway of the metamorphosis, can provide a theoretical basis for finding new targets of insecticides. This thesis aims to fly as the research object, analyzes 7 important genes of insulin signaling in the growth and development of B.dorsalis expression pattern period and body segments or tissues using RT-qPCR technology; analysis of the expression pattern of these gene in 20E and starvation stress stress; using RNAi technology to analysis the function of these genes; finally, using BdFoxO HPLC technology to detect silent dorsalis in the body of ecdysteroid titres influence, to further elucidate the association of insulin signaling pathway genes and ecdysone between. The results will clear the function of these genes and regulation, and provide ideas a new method for continuous control Bactrocera dorsalis. The main results are following: 1 on insulin signaling pathway Expression pattern analysis of 1.1 Bactrocera insulin signaling pathway expression patterns of 3 genes in different developmental stages of the insulin signaling pathway RT-qPCR technical analysis of 3 important genes of 3 genes (BdFoxO, BdFBP, BdPEPCK) expression patterns in various developmental stages on mRNA. The results showed that the relative expression of BdFoxO in larvae and pupae the amount is higher, and the highest expression in first day larvae after hatching; relative high expression of BdFBP in the larval stage, the differences of other developmental stages is not significant; BdPEPCK relative expression quantity in higher pupal stage, adult emergence after first days the expression was the highest. In combination with the results of previous study found that most of the insulin signaling pathway genes in the relative expression of larvae and pupae were higher, indicating the molting process of Bactrocera.1.2 insulin signaling genes involved in the regulation on these path 3 The expression pattern of genes in different tissues or body segments using the insulin signaling pathway RT-qPCR technical analysis for 3 genes (BdFoxO, BdFBP, BdPEPCK) in the chest, abdomen, its head, ovary, fat body midgut and Malpighian tubules, 7 different body segments or tissue mRNA expression patterns. The results showed that in bdfoxo the relative expression of the highest, followed by fat body.Bdfbp and bdpepck in the fat body of the highest expression level, and the expression of bdpepck in fat body weight is 102.1 times the amount of ovarian expression. The fat body can integrate a variety of nutritional and hormonal signals to control insects and individual cell size, insulin signaling pathway genes that these genes may influence further the growth process of.220e and starvation treatment on the expression of insulin signaling pathway genes of 7 Bactrocera 2.120e effect on orange in the dorsalis fat body high expression level relative The insulin signaling pathway arisanus 7 gene expression affected by microinjection of B.dorsalis fifth day old larvae were injected with different doses of 20E, through the detection of the insulin signaling pathway RT-qPCR 7 genes (Akt, PDK, pi3k21b, INR, bdfoxo, bdfbp and bdpepck) the relative expression level. The results showed that in addition to the relative expression of Akt in 20E after treatment significantly decreased, the other 6 genes were significantly up-regulated in general. It indicated that the exogenous hormone stimulated by insect insulin signaling gene transcription regulation and corresponding change, to achieve dynamic balance of each hormone body, so as to maintain the stability of its in vivo effect on the expression of.2.2 starvation the insulin signaling pathway in 7 genes using analytic Bactrocera RT-qPCR technology of Bactrocera dorsalis in insulin signaling pathway in 7 genes expression after starvation. Results show that, In addition to significant down-regulation of Akt and bdfbp in after starvation, significantly up-regulated expression genes. Most of the rest that larvae under starvation stress, may be coordinated through the insulin signaling pathway gene transcription level to strengthen the body function of signal transduction, so as to avoid the adverse environmental threat the continuation of Bactrocera.3 insulin signaling pathway 7 gene of its population to obtain high quality dsRNA by in vitro transcription, by microinjection of exogenous dsRNA into the 4 day old larvae of Bactrocera.Rt-qpcr analysis showed that 7 48h after gene silencing efficiency was about 50%, the highest silencing efficiency is above 80%. The expression level of 7 insulin signaling genes were detected there are different degrees of impact, found 7 genes between them, suggesting that these genes may exist between a series and negative feedback Silence after adjusting mechanism. The phenotypic observation of parasites, we find that most of the insulin signaling pathway gene silencing have a certain impact on the pupation time, that gene silencing and insulin signaling leads to some physiological changes related to the occurrence of Bactrocera dorsalis. Based on the injection of dsbdfoxo larvae was further observed that silencing of bdfoxo in larval weight significantly heavy body increases. These results further indicated that insulin signaling pathway genes on the growth and development of the important role of Bactrocera dorsalis.4 BdFoxO gene on the in vivo ecdysteroid titer detection of BdFoxO gene by using RNAi HPLC technology in Bactrocera ecdysteroid titers were observed. Results showed that the larvae after BdFoxO gene silencing, the in vivo 20E content this may be significantly decreased after BdFoxO gene silencing leads to B.dorsalis pupation The cause of the delay.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S433

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