本文關(guān)鍵詞：桔小實(shí)蠅胰島素信號(hào)通路基因表達(dá)特性及功能研究　出處：《西南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 桔小實(shí)蠅 胰島素信號(hào)通路 RT-qPCR RNAi HPLC

【摘要】：桔小實(shí)蠅Bactrocera dorsalis(Hendel)隸屬雙翅目Diptera、實(shí)蠅科Tephritidae,是一種廣泛分布于熱帶和亞熱帶地區(qū)的世界性農(nóng)業(yè)害蟲。該蟲寄主范圍廣、適應(yīng)能力強(qiáng)、繁殖力高且擴(kuò)散能力強(qiáng),是國(guó)際貿(mào)易的技術(shù)壁壘,被許多國(guó)家列為檢疫對(duì)象。桔小實(shí)蠅產(chǎn)卵于果實(shí)內(nèi),幼蟲潛居于果瓤中取食危害,導(dǎo)致果實(shí)腐爛、未熟先落,降低果蔬的經(jīng)濟(jì)價(jià)值和食用價(jià)值。目前,由于化學(xué)藥劑的不科學(xué)使用,桔小實(shí)蠅對(duì)多種常用殺蟲劑產(chǎn)生嚴(yán)重的抗藥性。因此,亟待尋找新的殺蟲劑作用靶標(biāo)或研發(fā)綠色防控技術(shù)。昆蟲胰島素信號(hào)的轉(zhuǎn)導(dǎo)能夠調(diào)節(jié)機(jī)體的生長(zhǎng)發(fā)育、代謝、生殖以及衰老等重要生理過(guò)程。因此,闡明桔小實(shí)蠅胰島素信號(hào)通路調(diào)控其變態(tài)發(fā)育的分子機(jī)制,可為尋找殺蟲劑新的作用靶標(biāo)提供理論基礎(chǔ)。本學(xué)位論文以桔小實(shí)蠅為研究對(duì)象,利用RT-qPCR技術(shù)分析胰島素信號(hào)通路7個(gè)重要基因在桔小實(shí)蠅各生長(zhǎng)發(fā)育時(shí)期和體段或組織中的表達(dá)模式;分析這些基因在20E和饑餓脅迫后的應(yīng)激表達(dá)模式;利用RNAi技術(shù)解析這些基因的功能;最后,利用HPLC技術(shù)檢測(cè)沉默桔小實(shí)蠅BdFoxO后對(duì)其體內(nèi)蛻皮激素滴度的影響,進(jìn)一步闡明胰島素信號(hào)通路基因與蛻皮激素間的關(guān)聯(lián)。研究結(jié)果將明確這些基因的功能以及調(diào)控方式,為桔小實(shí)蠅的持續(xù)防控提供新的思路和方法。主要研究結(jié)果如下:1桔小實(shí)蠅胰島素信號(hào)通路3個(gè)基因的時(shí)空表達(dá)模式分析1.1桔小實(shí)蠅胰島素信號(hào)通路3個(gè)基因在各發(fā)育時(shí)期的表達(dá)模式利用RT-qPCR技術(shù)解析胰島素信號(hào)通路3個(gè)重要基因(BdFoxO、BdFBP、BdPEPCK)在桔小實(shí)蠅的各發(fā)育時(shí)期的mRNA表達(dá)模式。結(jié)果表明,BdFoxO在幼蟲及蛹期的相對(duì)表達(dá)量較高,且最高表達(dá)量出現(xiàn)在幼蟲孵化后的第1天;BdFBP在幼蟲階段的相對(duì)表達(dá)量較高,其余發(fā)育時(shí)期間差異不顯著;BdPEPCK在蛹期相對(duì)表達(dá)量較高,成蟲羽化后第1天的表達(dá)量最高。結(jié)合前期研究結(jié)果發(fā)現(xiàn)大多數(shù)胰島素信號(hào)通路基因在桔小實(shí)蠅幼蟲及蛹期的相對(duì)表達(dá)量較高,說(shuō)明這些基因參與調(diào)控桔小實(shí)蠅的蛻皮過(guò)程。1.2桔小實(shí)蠅胰島素信號(hào)通路3個(gè)基因在不同體段或組織中的表達(dá)模式利用RT-qPCR技術(shù)解析胰島素信號(hào)通路3個(gè)重要基因(BdFoxO、BdFBP、BdPEPCK)在桔小實(shí)蠅頭部、胸部、腹部、卵巢、馬氏管、脂肪體及中腸7個(gè)不同體段或組織中的mrna表達(dá)模式。結(jié)果表明,bdfoxo在中腸相對(duì)表達(dá)量最高,其后為脂肪體。bdfbp和bdpepck在脂肪體中表達(dá)量最高,且bdpepck在脂肪體的表達(dá)量是卵巢表達(dá)量的102.1倍。脂肪體能夠整合多種營(yíng)養(yǎng)和激素信號(hào)調(diào)控昆蟲個(gè)體和細(xì)胞大小,胰島素信號(hào)通路基因在桔小實(shí)蠅脂肪體相對(duì)較高水平的表達(dá)進(jìn)一步說(shuō)明這些基因可能影響其生長(zhǎng)發(fā)育過(guò)程。220e和饑餓處理對(duì)桔小實(shí)蠅胰島素信號(hào)通路7個(gè)基因表達(dá)的影響2.120e處理對(duì)桔小實(shí)蠅胰島素信號(hào)通路7個(gè)基因表達(dá)的影響利用微量注射法,對(duì)桔小實(shí)蠅第5日齡幼蟲注射不同劑量的20e,通過(guò)rt-qpcr技術(shù)檢測(cè)胰島素信號(hào)通路7個(gè)基因(akt、pdk、pi3k21b、inr、bdfoxo、bdfbp以及bdpepck)的相對(duì)表達(dá)量。結(jié)果表明,除akt在20e處理后相對(duì)表達(dá)水平顯著下調(diào)外,其余6個(gè)基因總體呈顯著上調(diào)趨勢(shì)。說(shuō)明在外源激素的刺激下,昆蟲體內(nèi)胰島素信號(hào)通路基因轉(zhuǎn)錄水平出現(xiàn)相應(yīng)調(diào)節(jié)變化,使體內(nèi)各激素間達(dá)到動(dòng)態(tài)平衡,從而維持自身體內(nèi)的穩(wěn)定。2.2饑餓處理對(duì)桔小實(shí)蠅胰島素信號(hào)通路7個(gè)基因表達(dá)的影響利用rt-qpcr技術(shù)解析桔小實(shí)蠅胰島素信號(hào)通路7個(gè)基因在饑餓處理后的表達(dá)水平變化。結(jié)果表明,除akt及bdfbp在饑餓處理后顯著下調(diào)外,其余多數(shù)基因的表達(dá)顯著上調(diào)。推測(cè)桔小實(shí)蠅幼蟲在受到饑餓脅迫后,可能通過(guò)胰島素信號(hào)通路基因間轉(zhuǎn)錄水平的相互協(xié)調(diào)加強(qiáng)體內(nèi)信號(hào)轉(zhuǎn)導(dǎo),從而避免不良環(huán)境對(duì)自身種群延續(xù)產(chǎn)生的威脅。3桔小實(shí)蠅胰島素信號(hào)通路7個(gè)基因的功能初探通過(guò)體外轉(zhuǎn)錄法獲得高質(zhì)量dsrna,借助微量注射法將外源dsrna導(dǎo)入桔小實(shí)蠅4日齡幼蟲體內(nèi)。rt-qpcr分析結(jié)果表明,7個(gè)基因48h后沉默效率均在50%左右,沉默效率最高的在80%以上。分別檢測(cè)7個(gè)胰島素信號(hào)通路基因的表達(dá)水平,發(fā)現(xiàn)7個(gè)基因相互之間存在不同程度的影響,說(shuō)明這些基因之間可能存在一系列反饋與負(fù)反饋調(diào)節(jié)機(jī)制。沉默后對(duì)蟲體進(jìn)行表型觀察,發(fā)現(xiàn)大多胰島素信號(hào)通路基因沉默后對(duì)其幼蟲化蛹時(shí)間產(chǎn)生一定的影響,推測(cè)與胰島素信號(hào)通路的基因沉默導(dǎo)致桔小實(shí)蠅發(fā)生某些生理變化有關(guān)。通過(guò)對(duì)注射dsbdfoxo的幼蟲進(jìn)一步觀察發(fā)現(xiàn),沉默bdfoxo的幼蟲體重明顯變重,蟲體增大。這一結(jié)果進(jìn)一步說(shuō)明胰島素信號(hào)通路基因?qū)坌?shí)蠅生長(zhǎng)發(fā)育具有重要作用。4桔小實(shí)蠅BdFoxO基因?qū)ζ潴w內(nèi)蛻皮激素滴度的影響利用HPLC技術(shù)檢測(cè)BdFoxO基因RNAi后桔小實(shí)蠅體內(nèi)蛻皮激素滴度的變化情況。結(jié)果發(fā)現(xiàn),桔小實(shí)蠅幼蟲BdFoxO基因沉默后,其體內(nèi)20E含量極顯著降低,這可能是桔小實(shí)蠅BdFoxO基因沉默后導(dǎo)致化蛹時(shí)間延遲的原因。
[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.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S433

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