本文選題：桔小實蠅　切入點：酚氧化酶　出處：《西南大學》2015年碩士論文　論文類型：學位論文

【摘要】：酚氧化酶在昆蟲的生長發(fā)育和變態(tài)過程中具有重要作用,該酶主要參與昆蟲傷口愈合、黑化作用和硬化作用。其中,黑化與硬化在昆蟲表皮的形成、抵御外源病原菌以及免疫反應中起到重要的作用。因此,酚氧化酶及其酶原蛋白成為昆蟲生長發(fā)育與免疫反應研究的熱點。桔小實蠅是一種極具破壞力的多食性害蟲,可危害250多種果蔬。化蛹和羽化是昆蟲在生長發(fā)育過程中,面臨復雜環(huán)境而成功生存繁衍的重要變態(tài)過程。桔小實蠅成蟲產(chǎn)卵于果實中,幼蟲期主要在果實內(nèi)部為害,造成果實腐爛落地,進而在土壤中化蛹羽化為成蟲后繼續(xù)為害。當前,對桔小實蠅的防治主要依靠化學藥劑,導致桔小實蠅對多種化學藥劑產(chǎn)生了抗性,所以尋找新方法新途徑進行治理已刻不容緩。本學位論文研究以桔小實蠅為對象,以酚氧化酶及其酶原為靶標,針對桔小實蠅幼蟲-蛹變態(tài)階段該酶的作用開展了系統(tǒng)而深入地研究,主要結(jié)果如下：1.桔小實蠅不同發(fā)育階段及組織中酚氧化酶酶學特性及其酶原基因表達模式以鄰苯二酚為底物,分別測定桔小實蠅不同發(fā)育階段酚氧化酶活力。結(jié)果表明：桔小實蠅在不同發(fā)育階段酚氧化酶活力的高低存在顯著性差異,其中3齡幼蟲的酶活力最高,1齡幼蟲的酶活力最低。測定桔小實蠅3齡幼蟲表皮、脂肪體和中腸等組織的酚氧化酶活力,結(jié)果表明：桔小實蠅表皮、脂肪體和中腸等組織均可檢測到酚氧化酶的活性。其中表皮中酚氧化酶活力最高,而脂肪體中酚氧化酶活力最低。分析桔小實蠅酚氧化酶的動力學常數(shù)發(fā)現(xiàn),以鄰苯二酚為底物時,桔小實蠅不同發(fā)育階段間酚氧化酶的米氏常數(shù)(Km)值差異顯著,1齡、2齡幼蟲中酚氧化酶的最大反應速度(Vmax)值顯著大于3齡幼蟲、蛹和成蟲的Vmax。以L-多巴為底物結(jié)果大致相同。桔小實蠅不同組織間酚氧化酶的Km值無顯著性差異,表皮酚氧化酶的Vmax值顯著高于脂肪體和中腸酚氧化酶的Vmax值；但以L-多巴為底物時,各組織酚氧化酶的Km和Vmax均無顯著性差異。桔小實蠅不同發(fā)育階段及組織中酚氧化酶酶促反應的最適pH為7.5,溫度為37。C。采用qPCR技術,以α-Tubulin為內(nèi)參基因,解析桔小實蠅酚氧化酶酶原基因(BdPPO1)在不同發(fā)育階段及組織的表達模式。結(jié)果表明,BdPPO1主要在桔小實蠅幼蟲-蛹轉(zhuǎn)化期以及表皮中表達量較高,說明酚氧化酶活力及BdPPO1表達量與桔小實蠅生長發(fā)育緊密相關,尤其是在其變態(tài)發(fā)育階段。2.曲酸對桔小實蠅酚氧化酶及生長發(fā)育的影響曲酸是酚氧化酶的典型抑制劑,能夠有效的抑制昆蟲酚氧化酶的活性。將曲酸加入到桔小實蠅人工飼料并喂食新孵化的幼蟲,結(jié)果發(fā)現(xiàn),桔小實蠅幼蟲期和蛹期顯著延長,化蛹率和羽化率顯著降低,幼蟲體態(tài)明顯小于對照組。以鄰苯二酚為底物,喂食曲酸飼料的桔小實蠅幼蟲體內(nèi)及表皮中酚氧化酶酶活力顯著降低,而BdPPO1基因表達量顯著上調(diào),推測桔小實蠅體內(nèi)可能存在一種自我補償機制。進一步分析酚氧化酶動力學發(fā)現(xiàn),飼喂曲酸后酚氧化酶的Km值未發(fā)生顯著變化,但Vmax值顯著降低,酶促反應的最適pH和溫度未發(fā)生變化。上述結(jié)果說明曲酸能夠有效抑制桔小實蠅酚氧化酶的活性,并且導致桔小實蠅生長發(fā)育延遲。3.20-羥基蛻皮酮(20E)及金屬離子對桔小實蠅BdPPO1表達量的影響對桔小實蠅3齡幼蟲注射20E后,發(fā)現(xiàn)桔小實蠅幼蟲提前化蛹。檢測注射不同濃度的20E后BdPPO1的表達量發(fā)現(xiàn),與對照相比,注射濃度為0.5μg/mL時該基因表達量顯著上調(diào),而注射濃度降低或提高至0.15μg/mL和15μg/mL時該基因的表達量顯著下調(diào)。離體酶活性測定發(fā)現(xiàn),Zn2+、Mg2+、Ca2+和Cu2+等4種二價金屬離子均能有效地激活并提高桔小實蠅酚氧化酶的活力。向新孵化幼蟲飼喂含金屬離子的人工飼料6d后,酚氧化酶活力顯著升高,其中飼喂鋅離子后酚氧化酶活力最高,而飼喂鎂離子后酚氧化酶活力最低。進一步采用qPCR技術檢測桔小實蠅BdPPO1表達量發(fā)現(xiàn),飼喂4種金屬離子后該基因的表達量均顯著上調(diào)。上述結(jié)果說明,桔小實蠅BdPPO1表達量受20E調(diào)控,酚氧化酶作為一種金屬酶,能夠有效的被金屬離子激活。4.桔小實蠅BdPPO1基因的功能驗證利用RNAi技術進一步驗證了桔小實蠅BdPPO1基因的功能。結(jié)果表明,對桔小實蠅3齡幼蟲注射BdPPO1基因dsRNA 24 h和48 h后,BdPPO1基因達量顯著降低,幼蟲出現(xiàn)不能正�；�、表皮黑化等現(xiàn)象,同時化蛹率顯著降低。上述結(jié)果說明BdPPO1基因可作為一個潛在的靶標來開發(fā)新的防治藥劑,特別是具有作為基于RNAi技術防控桔小實蠅及其它害蟲的潛力。
[Abstract]:Phenoloxidase plays an important role in the growth and metamorphosis of insects, the main enzymes involved in insect wound healing, melanization and hardening effect. Among them, the formation of blackening and hardening in insect cuticle, resist play an important role of exogenous pathogenic bacteria and the immune response. Therefore, phenol oxidase and its proenzyme protein has become a hot spot insect growth and immune response studies. Dorsalis is a highly destructive polyphagous pest can harm more than 250 kinds of fruits and vegetables. Pupation and eclosion of insects in the growth process, facing the complex environment and important metamorphosis success. The survival of adultb eggs in fruit, larvae mainly inside the fruit damage caused by the rotten fruit fall, then pupate in soil after emergence as adults continue to damage. At present, the prevention and control of B.dorsalis mainly rely on chemicals, lead Fly produced resistance to various chemicals, so looking for a new way of governance has been urgent. The research of this thesis is to fly as the object, with PO and propo as a target for the larval pupal metamorphosis of the Bactrocera enzyme development system and in-depth research, the main results are as follows: phenol oxidase the characteristics of 1. different developmental stages and tissues of Bactrocera dorsalis and prophenoloxidase gene expression patterns with catechol as substrate, its different developmental stages of phenoloxidase activity were measured. The results showed that there were significant differences of B.dorsalis in different developmental stages of the phenoloxidase activity level of the enzyme activity of 3 instar larvae of 1 instar larvae of the highest enzyme activity was the lowest. Determination of 3 instar larvae of Bactrocera epidermis, phenoloxidase activity in midgut and fat body tissues. The results showed that: Orange Small fruit epidermis, midgut and fat body tissues can be detected in phenol oxidase activity. The phenoloxidase activity in the epidermis of the highest, and the lowest phenoloxidase activity in the fat body. Analysis of the kinetic constants of Bactrocera dorsalis phenoloxidase found with catechol as substrate, the Michaelis constant of Bactrocera dorsalis in different developmental stages between the phenol oxidase (Km) is significantly different between the age of 1, the maximum reaction rate of phenol oxidase in 2 instar larvae (Vmax) was significantly greater than 3 instar larvae, pupae and adults of Vmax. L- with DOPA as substrate were almost the same. Different groups of ORIMA phenol oxidase Bactrocera Km value was no significant difference in cuticular phenoloxidase Vmax the value was significantly higher than that of midgut and fat body of phenol oxidase Vmax; but L- DOPA as substrate, there were no significant differences in the organization of Km and Vmax of phenol oxidase. The phenol oxidation in different developmental stages and tissues of Bactrocera dorsalis The optimum pH of enzyme reaction was 7.5, temperature of 37.C. using qPCR technology, a -Tubulin as reference gene, analysis of Bactrocera phenoloxidase (BdPPO1) prothrombin gene expression patterns in different developmental stages and tissues. The results showed that BdPPO1 mainly in the larvae pupae transformation period and higher expression in the epidermis. That expression of phenoloxidase activity and the amount of BdPPO1 and its growth and development are closely related, especially in the metamorphosis of.2. effects on phenol oxidase and kojic acid dorsalis growth inhibitor is a typical kojic acid phenol oxidase, can effectively inhibit the phenoloxidase activity. The kojic acid added to the artificial diet and feeding Bactrocera newly hatched larvae the results showed that, significantly prolonged fly larvae and pupae, pupation rate and eclosion rate of larvae was significantly reduced, significantly less than the control group. The adjacent benzene two Phenol as substrate, phenoloxidase activity of enzyme in vivo and feeding larvae feed in the epidermis of kojic acid decreased significantly, while the expression of BdPPO1 gene was up-regulated in vivo, speculated that there may be a fly self compensation mechanism. Further analysis found that the kinetics of phenol oxidase, kojic acid and phenol oxidase after feeding Km value did not change significantly, but the Vmax value significantly reduced enzymatic reaction and the optimum pH and temperature did not change. The above results show that kojic acid can effectively inhibit its phenol oxidase activity and lead to delayed growth.3.20- dorsalis HYDROXYECDYSONE (20E) affect the amount of fruit and metal ions on the expression of BdPPO1 3 instar larvae after injection of 20E, found that the larvae in advance pupation. Different concentrations of 20E after injection to detect the expression of BdPPO1 was found, compared with the control, injection concentration is 0.5 g/mL The gene expression was up-regulated, and the injection concentration decrease or increase to the gene expression was significantly reduced by 0.15 g/mL and 15 g/mL. In vitro enzyme activity assay showed that Zn2+, Mg2+, Ca2+ and Cu2+ 4 kinds of two valence metal ions can effectively activate and improve phenol oxidizing enzyme activity of Bactrocera dorsalis 6D. Artificial feed to new hatching larvae were fed a diet containing metal ions, phenoloxidase activity increased significantly, which fed the zinc ions after phenol oxidase activity is the highest, while feeding the magnesium ions after phenol oxidase activity was the lowest. Further qPCR was used to detect the expression of Bactrocera BdPPO1 found that feeding 4 kinds of metal ions after the gene expression quantity increased significantly. The results showed that the expression of BdPPO1 regulated by 20E dorsalis, phenol oxidase as a metal enzyme, can effectively be metal ion activated.4. dorsalis BdPPO1 gene function verification and Using RNAi technology to further validate the BdPPO1 gene function. The results show that on the fly 3 instar larvae injected with BdPPO1 gene dsRNA 24 h and 48 h, the BdPPO1 gene was significantly reduced and the larvae cannot pupate, skin blackening phenomenon, but the pupation rate significantly decreased. These results showed that BdPPO1 gene could be used as a a potential target for the development of new insecticides, especially with RNAi technology as control B.dorsalis and other pests based on the potential.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S433

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