桔小实蝇酚氧化酶酶学特性及其酶原基因的研究

发布时间：2018-03-19 12:50

本文选题：桔小实蝇　切入点：酚氧化酶　出处：《西南大学》2015年硕士论文　论文类型：学位论文

【摘要】：酚氧化酶在昆虫的生长发育和变态过程中具有重要作用,该酶主要参与昆虫伤口愈合、黑化作用和硬化作用。其中,黑化与硬化在昆虫表皮的形成、抵御外源病原菌以及免疫反应中起到重要的作用。因此,酚氧化酶及其酶原蛋白成为昆虫生长发育与免疫反应研究的热点。桔小实蝇是一种极具破坏力的多食性害虫,可危害250多种果蔬。化蛹和羽化是昆虫在生长发育过程中,面临复杂环境而成功生存繁衍的重要变态过程。桔小实蝇成虫产卵于果实中,幼虫期主要在果实内部为害,造成果实腐烂落地,进而在土壤中化蛹羽化为成虫后继续为害。当前,对桔小实蝇的防治主要依靠化学药剂,导致桔小实蝇对多种化学药剂产生了抗性,所以寻找新方法新途径进行治理已刻不容缓。本学位论文研究以桔小实蝇为对象,以酚氧化酶及其酶原为靶标,针对桔小实蝇幼虫-蛹变态阶段该酶的作用开展了系统而深入地研究,主要结果如下：1.桔小实蝇不同发育阶段及组织中酚氧化酶酶学特性及其酶原基因表达模式以邻苯二酚为底物,分别测定桔小实蝇不同发育阶段酚氧化酶活力。结果表明：桔小实蝇在不同发育阶段酚氧化酶活力的高低存在显著性差异,其中3龄幼虫的酶活力最高,1龄幼虫的酶活力最低。测定桔小实蝇3龄幼虫表皮、脂肪体和中肠等组织的酚氧化酶活力,结果表明：桔小实蝇表皮、脂肪体和中肠等组织均可检测到酚氧化酶的活性。其中表皮中酚氧化酶活力最高,而脂肪体中酚氧化酶活力最低。分析桔小实蝇酚氧化酶的动力学常数发现,以邻苯二酚为底物时,桔小实蝇不同发育阶段间酚氧化酶的米氏常数(Km)值差异显著,1龄、2龄幼虫中酚氧化酶的最大反应速度(Vmax)值显著大于3龄幼虫、蛹和成虫的Vmax。以L-多巴为底物结果大致相同。桔小实蝇不同组织间酚氧化酶的Km值无显著性差异,表皮酚氧化酶的Vmax值显著高于脂肪体和中肠酚氧化酶的Vmax值；但以L-多巴为底物时,各组织酚氧化酶的Km和Vmax均无显著性差异。桔小实蝇不同发育阶段及组织中酚氧化酶酶促反应的最适pH为7.5,温度为37。C。采用qPCR技术,以α-Tubulin为内参基因,解析桔小实蝇酚氧化酶酶原基因(BdPPO1)在不同发育阶段及组织的表达模式。结果表明,BdPPO1主要在桔小实蝇幼虫-蛹转化期以及表皮中表达量较高,说明酚氧化酶活力及BdPPO1表达量与桔小实蝇生长发育紧密相关,尤其是在其变态发育阶段。2.曲酸对桔小实蝇酚氧化酶及生长发育的影响曲酸是酚氧化酶的典型抑制剂,能够有效的抑制昆虫酚氧化酶的活性。将曲酸加入到桔小实蝇人工饲料并喂食新孵化的幼虫,结果发现,桔小实蝇幼虫期和蛹期显著延长,化蛹率和羽化率显著降低,幼虫体态明显小于对照组。以邻苯二酚为底物,喂食曲酸饲料的桔小实蝇幼虫体内及表皮中酚氧化酶酶活力显著降低,而BdPPO1基因表达量显著上调,推测桔小实蝇体内可能存在一种自我补偿机制。进一步分析酚氧化酶动力学发现,饲喂曲酸后酚氧化酶的Km值未发生显著变化,但Vmax值显著降低,酶促反应的最适pH和温度未发生变化。上述结果说明曲酸能够有效抑制桔小实蝇酚氧化酶的活性,并且导致桔小实蝇生长发育延迟。3.20-羟基蜕皮酮(20E)及金属离子对桔小实蝇BdPPO1表达量的影响对桔小实蝇3龄幼虫注射20E后,发现桔小实蝇幼虫提前化蛹。检测注射不同浓度的20E后BdPPO1的表达量发现,与对照相比,注射浓度为0.5μg/mL时该基因表达量显著上调,而注射浓度降低或提高至0.15μg/mL和15μg/mL时该基因的表达量显著下调。离体酶活性测定发现,Zn2+、Mg2+、Ca2+和Cu2+等4种二价金属离子均能有效地激活并提高桔小实蝇酚氧化酶的活力。向新孵化幼虫饲喂含金属离子的人工饲料6d后,酚氧化酶活力显著升高,其中饲喂锌离子后酚氧化酶活力最高,而饲喂镁离子后酚氧化酶活力最低。进一步采用qPCR技术检测桔小实蝇BdPPO1表达量发现,饲喂4种金属离子后该基因的表达量均显著上调。上述结果说明,桔小实蝇BdPPO1表达量受20E调控,酚氧化酶作为一种金属酶,能够有效的被金属离子激活。4.桔小实蝇BdPPO1基因的功能验证利用RNAi技术进一步验证了桔小实蝇BdPPO1基因的功能。结果表明,对桔小实蝇3龄幼虫注射BdPPO1基因dsRNA 24 h和48 h后,BdPPO1基因达量显著降低,幼虫出现不能正常化蛹、表皮黑化等现象,同时化蛹率显著降低。上述结果说明BdPPO1基因可作为一个潜在的靶标来开发新的防治药剂,特别是具有作为基于RNAi技术防控桔小实蝇及其它害虫的潜力。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S433

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