本文選題：桔小實蠅　切入點(diǎn)：小分子熱激蛋白　出處：《西南大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：桔小實蠅Bactrocera dorsalis (Hendel)隸屬雙翅目Diptera、實蠅科Tephritidae,廣泛分布于熱帶和亞熱帶地區(qū)。桔小實蠅寄主范圍廣、適應(yīng)性、繁殖力以及擴(kuò)散能力強(qiáng),是一種重要的農(nóng)業(yè)害蟲。桔小實蠅的防治目前主要是依靠化學(xué)藥劑,由于大量不合理地使用化學(xué)殺蟲劑,造成了桔小實蠅對多種常用殺蟲劑產(chǎn)生了嚴(yán)重的抗藥性。因此,尋找新的防治突破點(diǎn)顯得尤為重要。昆蟲小分子熱激蛋白可以參與調(diào)節(jié)機(jī)體的生長發(fā)育、抗逆、生殖以及細(xì)胞分化等重要生理過程。因此,闡明桔小實蠅小分子熱激蛋白調(diào)控其變態(tài)發(fā)育和抗逆過程中的分子機(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)位論文基于桔小實蠅轉(zhuǎn)錄組數(shù)據(jù)庫信息,運(yùn)用RT-PCR技術(shù),從桔小實蠅體內(nèi)克隆獲得了9個小分子熱激蛋白(small heat shock protein, sHSP)基因的cDNA序列,并對其特征序列進(jìn)行注釋；利用qPCR技術(shù)解析了這9個基因在桔小實蠅不同發(fā)育階段和成蟲不同組織的表達(dá)特性,以及外源20E和熱脅迫后的應(yīng)激表達(dá)模式；最后利用RNAi技術(shù)探索了小分子熱激蛋白基因Bdhsp23.8和3315在桔小實蠅卵巢發(fā)育中的作用,驗證了小分子熱激蛋白基因Bdhsp23.8和Bdhsp20.6在桔小實蠅抗熱脅迫中的作用。研究結(jié)果將有助于解析桔小實蠅小分子熱激蛋白基因的功能及其調(diào)控機(jī)制,為桔小實蠅的持續(xù)防控提供新的思路和方法。主要研究結(jié)果如下：1桔小實蠅9個sHSP基因的克隆與序列分析根據(jù)從桔小實蠅轉(zhuǎn)錄組數(shù)據(jù)庫篩選鑒定的unigene序列,克隆獲得了桔小實蠅9個sHSP基因的cDNA序列,這9個基因分別為Bdhsp20.4、Bdhsp21.6、 Bdhsp23.8、Bdhsp20.6、Bdhsp23.0、Bdhsp17.6、Bdhsp18.4、Bdhsp11.1和3315。通過序列分析,明確了其中8個基因的開放閱讀框,并推導(dǎo)了其編碼的氨基酸序列。2桔小實蠅9個sHSP基因的表達(dá)模式分析2.1桔小實蠅9個sHSP基因在不同發(fā)育階段的表達(dá)模式利用qPCR技術(shù)分析了桔小實蠅9個sHSP基因在卵、幼蟲、蛹和成蟲期的mRNA表達(dá)模式。結(jié)果表明,這9個基因在桔小實蠅不同發(fā)育階段的表達(dá)量差異明顯,其中Bdhsp20.4和Bdhsp18.4在卵、幼蟲和蛹前期的相對表達(dá)非常低,在蛹的后期和成蟲期的相對表達(dá)量激增；Bdhsp23.8和Bdhsp23.0在卵、幼蟲和蛹前期的相對表達(dá)量明顯高于幼蟲后期和蛹后期；Bdhsp21.6在卵期幾乎不表達(dá),在幼蟲和蛹期的相對表達(dá)變化非常明顯；Bdhsp17.6在卵中幾乎不表達(dá),在幼蟲中的表達(dá)非常高,并且前期高于后期,蛹中則相反,隨著日齡的增加,表達(dá)量逐漸升高。2.2桔小實蠅9個sHSP基因在成蟲不同組織中的表達(dá)模式利用qPCR技術(shù)解析了桔小實蠅9個sHSP基因在成蟲頭、胸、中腸、馬氏管、脂肪體、精巢和卵巢中的相對表達(dá)量。結(jié)果表明,Bdhsp23.8和3315在卵巢中的相對表達(dá)量顯著高于其他組織,由此可推測Bdhsp23.8和3315在桔小實蠅的卵巢發(fā)育中起到了不可缺少的作用；Bdhsp21.6、Bdhsp23.0、Bdhsp20.6和Bdhsp11.1在各組織中的相對表達(dá)量也有顯著性差異；Bdhsp20.4在脂肪體中的表達(dá)量顯著高于其他組織,由此可初步推斷該基因和桔小實蠅的能量代謝相關(guān)；Bdhsp17.6在頭、胸和脂肪體中相對表達(dá)量顯著高于其他組織,在精巢和卵巢中不表達(dá)；Bdhsp18.4僅在桔小實蠅的胸部高表達(dá),其他組織中幾乎不表達(dá)。3外源20E和熱脅迫對9個sHSP基因表達(dá)的影響3.1 20E對9個sHSP基因表達(dá)的影響用不同劑量的20E處理桔小實蠅5日齡幼蟲,結(jié)果表明,注射1000ng的20E后12h,Bdhsp20.4、Bdhsp21.6、Bdhsp23.8、Bdhsp23.0、Bdhsp20.6、Bdhsp18.4和3315的相對表達(dá)量均顯著上調(diào),Bdhsp11.1明顯下調(diào),Bdhsp17.6則沒有顯著變化。3.2熱脅迫對9個sHSP基因表達(dá)的影響用不同溫度處理桔小實蠅7日齡成蟲,結(jié)果表明,Bdhsp20.4和Bdhsp21.6的相對表達(dá)量僅在-5℃脅迫下顯著上調(diào)；Bdhsp23.8、Bdhsp20.6和Bdhsp18.4在-5℃和40℃脅迫下均顯著上調(diào)；而Bdhsp17.6的相對表達(dá)量不會受溫度的影響；Bdhsp11.1的相對表達(dá)量經(jīng)-5℃和0℃脅迫后顯著下調(diào)。4桔小實蠅Bdhsp23.8、Bdhsp20.6和3315基因的功能研究將體外合成的Bdhsp23.8和3315基因的dsRNA注射入未交配的桔小實蠅4日齡雌成蟲體內(nèi),并在72h后解剖其卵巢,在體視鏡下觀察卵巢的發(fā)育情況,同時利用qPCR技術(shù)檢測目的基因的沉默效率。結(jié)果顯示,其mRNA的相對表達(dá)明顯下調(diào),且卵巢的發(fā)育較對照明顯滯后。將體外合成的Bdhsp23.8和Bdhsp20.6基因的dsRNA注射入桔小實蠅7日齡成蟲體內(nèi),經(jīng)-5℃和40℃脅迫,觀察桔小實蠅的死亡率,并利用qPCR技術(shù)檢測目的基因的沉默效率。結(jié)果表明,干擾Bdhsp20.6基因12h后經(jīng)-5℃和40℃脅迫,桔小實蠅死亡率顯著高于對照組,mRNA的相對表達(dá)明顯下調(diào)；然而48h后,桔小實蠅的死亡率和mRNA的沉默效率都出現(xiàn)大幅度降低。注射dsBdhsp23.8后,桔小實蠅幾乎沒有死亡,并且mRNA的相對表達(dá)也無明顯下調(diào),檢測Bdhsp20.6基因的相對表達(dá)量,結(jié)果顯示也沒有明顯下調(diào),對Bdhsp23.8溫度相關(guān)功能的RNAi仍需進(jìn)一步探索。
[Abstract]:Bactrocera dorsalis (Hendel) Diptera belongs to Diptera, Tephritidae Tephritidae, widely distributed in tropical and subtropical regions. Its wide host range, adaptability, fecundity and diffusion ability, is an important agricultural pest control. Its currently relies mainly on chemical agents, due to the large number of unreasonable use of chemical pesticides. Which fruit developed serious resistance to various insecticides. Therefore, to find a new breakthrough point of prevention is particularly important. The insect sHSP can participate and regulate the body's growth and development, reproduction and resistance, cell differentiation and other physiological processes. Therefore, to clarify the B.dorsalis small heat shock protein regulates the metamorphosis and molecular mechanisms of stress resistance in the process, can provide a theoretical basis for finding new targets of insecticides. This paper base In the Bactrocera dorsalis transcriptome database, using RT-PCR technology, 9 small heat shock protein obtained from in vivo cloning (small heat shock on protein, sHSP) cDNA gene sequences, and comments on its characteristic sequence; using qPCR technology to analysis the 9 gene expression in different developmental characteristics of oriental fruit fly the stage and adult tissues, and exogenous 20E and heat stress after the stress expression pattern; finally explores the small heat shock protein gene Bdhsp23.8 and 3315 in the role of B.dorsalis ovarian development by using RNAi technology, verifies the effect of small heat shock protein genes Bdhsp23.8 and Bdhsp20.6 in its thermal stress. The results will be in have the function and regulatory mechanism contributes to the analysis of Bactrocera dorsalis small heat shock protein gene, provide new ideas and methods for the prevention and control of continuous. The main research fruit The results are as follows: 9 cloning and sequence analysis of sHSP gene according to the sequence of UniGene 1 Bactrocera dorsalis from the screening and identification of transcriptome database, cloned the cDNA sequence of 9 sHSP genes on, these 9 genes were Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp20.6, Bdhsp23.0, Bdhsp17.6, Bdhsp18.4, Bdhsp11.1 and 3315. by sequence analysis clearly, the 8 gene open reading frame, and deduced 2.1 analysis of 9 sHSP genes of Bactrocera dorsalis larvae of 9 sHSP genes in oocytes at different developmental stages, the expression patterns by qPCR expression patterns of 9 sHSP genes encoding the amino acid sequence of.2 dorsalis, pupa and adult stage the expression pattern of mRNA. The results showed that the expression of these 9 genes in different developmental stages of B.dorsalis was different, in which Bdhsp20.4 and Bdhsp18.4 in eggs, larvae and pupae before The relative expression is very low, the relative expression of late pupal and adult stages of the surge; Bdhsp23.8 and Bdhsp23.0 in eggs, larvae and pupae of the relative expression was significantly higher than that of larvae and pupae of the late late; Bdhsp21.6 almost no expression in the egg stage, very obvious changes in the relative scale of larval and pupal stages; Bdhsp17.6 almost no expression in eggs, expressed in larvae is very high, and higher than the pre pupa stage, on the contrary, with the increase of age, the expression of.2.2 gradually increased 9 Bactrocera sHSP gene in different tissues of adult expression pattern in the use of qPCR to analysis of fruit 9 sHSP genes in the adult head. The chest, midgut, Malpighian tubules, fat body, the relative expression in the testis and ovary. The results showed that Bdhsp23.8 and 3315 in the ovary of the relative expression was significantly higher than that in other tissues, which can be speculated that Bdhsp23.8 and 3315 in small orange Tephritidae ovarian development plays an indispensable role; Bdhsp21.6, Bdhsp23.0, Bdhsp20.6 and Bdhsp11.1 in tissues of relative expression also have significant differences; the expression of Bdhsp20.4 in fat body weight was significantly higher than that in other tissues, thus it is inferred that the energy metabolism related genes and Bactrocera Bdhsp17.6 in head,; the chest fat body and the relative expression level was significantly higher than that in other tissues, no expression in testis and ovary; Bdhsp18.4 high expression only in on chest, affect the other tissues almost does not affect the expression of.3 of exogenous 20E and heat stress on the expression of 9 sHSP genes of 3.1 20E on the expression of 9 sHSP genes by 20E treatment of Bactrocera dorsalis different doses of 5 day old larvae, the results show that the injection of 1000ng 20E 12h, Bdhsp20.4, Bdhsp21.6, Bdhsp23.8, Bdhsp23.0, Bdhsp20.6, Bdhsp18.4 and 3315 relative expression were significantly. Bdhsp11.1, the down-regulation of Bdhsp17.6 is not significantly affected the change of.3.2 heat stress on the expression of 9 sHSP genes with different temperature of Bactrocera dorsalis 7 day old adults. The results showed that the relative expression of Bdhsp20.4 and Bdhsp21.6 only at -5 DEG C stress significantly increased; Bdhsp23.8, Bdhsp20.6 and Bdhsp18.4 at -5 DEG C and 40 DEG C under stress windgroup; Bdhsp17.6 relative expression quantity is not affected by the temperature effect; relative expression of Bdhsp11.1 by -5 stress degrees and 0 degrees after significant downregulation of.4 Bdhsp23.8, function of Bdhsp20.6 and Bdhsp23.8 3315 gene synthesis in vitro and 3315 gene dsRNA was injected into virgin fly 4 day old female adults in vivo, and the ovarian anatomy in 72h after ovarian development was observed under optical microscopy, and using qPCR technology to detect gene silencing efficiency. The results showed that the mRNA phase The expression was downregulated, and ovarian development is lagging behind. The lighting of Bdhsp23.8 and Bdhsp20.6 gene in vitro synthesis of dsRNA injected into the fly 7 day old adults in the -5 degrees and 40 degrees of stress, the observation of B.dorsalis mortality, and the use of qPCR technology to examine the gene silencing efficiency. The results show that the interference of Bdhsp20.6 gene 12h after -5 degrees and 40 degrees of stress, its mortality rate was significantly higher than the control group, the relative expression of mRNA was significantly reduced after 48h; however, its mortality and silencing efficiency of mRNA are greatly reduced. After injection of dsBdhsp23.8, mRNA and Bactrocera almost no death, the relative expression was significantly reduced without relative expression Bdhsp20.6 gene, showed no obvious reduction, the temperature of the Bdhsp23.8 function of RNAi still need further exploration.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S433;Q78

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