光酶诱导桑叶中桑辛素N抗BmNPV机制及其生物合成关键酶的研究

发布时间：2018-09-01 17:03

【摘要】：家蚕(Bombyx mori L.)属鳞翅目、蚕蛾科,家蚕核型多角体病毒(BmNPV)病是家蚕饲养过程中最常见、最严重的一类蚕病,造成了蚕桑业巨大的经济损失。目前,针对家蚕BmNPV病仍然没有有效的治疗方法,其抗病机制尚不清楚。桑叶是家蚕的主要食物来源,也是一种重要的药用植物。前期研究发现,桑叶经UV-B诱导之后,次生代谢产物的含量发生变化,其中桑辛素N的含量显著增加。本文首次发现饲喂UV-B诱导的桑叶能提高家蚕对于BmNPV病的抗性,并推测桑辛素N是其主要的有效成分。利用label-free技术对家蚕中肠蛋白进行差异蛋白质组学分析,揭示了诱导桑叶及桑辛素N的抗病机制。研究结果为家蚕核型多角体病的治疗提供了新的方案,也为之后的研究奠定了基础。此外,对诱导桑叶中桑辛素N的生物合成关键酶进行了研究,发现诱导桑叶中存在一种新型氧化环合酶,能催化氧化白藜芦醇环合生成桑辛素N的重要前体——桑辛素M。该研究结果为探讨UV-B诱导桑叶中次生代谢产物的变化奠定了基础。本文的主要研究内容和结果如下:.(1)光酶诱导桑叶中桑辛素N抗BmNPV的机制研究BmNPV是对家蚕造成危害最大的病毒之一,家蚕在患BmNPV病后期常流出脓汁,内含大量的多角体病毒,俗称脓病。这类疾病造成家蚕死亡,导致了蚕桑业的巨大经济损失。家蚕抗病毒实验发现饲养UV-B诱导的桑叶或涂有桑辛素N的桑叶之后,家蚕对BmNPV病的抗性得到提高,感染病毒之后死亡率降低。为了进一步研究其抗病机制,分别取饲喂诱导桑叶(BUM)与桑辛素N(BNM)的家蚕中肠进行比较蛋白质组学分析,结果在BUM组与BNM组中分别鉴定到413和102个差异蛋白。利用生物信息学进一步分析,发现诱导桑叶和桑辛素N可使较多的核糖体蛋白表达变化,调控蛋白的合成、DNA的修复;可以使ATP酶表达上调、ATP合成酶表达下调,以此改变细胞内环境来阻止病毒脱壳进去细胞核,抵抗病毒的侵染。此外,桑辛素N还可以使细胞色素氧化酶、Ca2+相关蛋白与programmed cell death protein表达上调从而促进细胞凋亡,抑制病毒扩散与复制;可以使lipase-1与serine protease precursor表达上调,来增强家蚕机体免疫力,抵抗病毒入侵;可以使组蛋白、Rab蛋白表达上调,前者可与病毒结合,后者可作为信号分子,在家蚕抗BmNPV过程中起到特殊作用。以上结果揭示了诱导桑叶和桑辛素N的抗病过程,这对于提高感染BmNPV家蚕的存活率是一个新的治疗方案。(2)诱导桑叶中桑辛素N生物合成关键酶的研究桑(Morus alba L.),桑科桑属,落叶乔木或灌木,是一种重要的药用植物,具有多种药理作用,也是家蚕的主要食物来源。桑辛素是桑叶中重要的次生代谢产物,具有抗肿瘤、抗炎、抗病毒等活性。桑叶经UV-B诱导,次生代谢产物的含量发生变化,其中桑辛素N的含量显著增加。目前,关于桑辛素N的化学合成已经有报道,但是其生物合成至今无详细的报道。课题组前期研究工作发现桑辛素N生物合成的重要前体是桑辛素M,本文首次发现了诱导桑叶中存在一种新型桑辛素氧化环合酶能催化氧化白藜芦醇环合生成桑辛素M,这对于阐明桑辛素N的生物合成具有重要意义。此外,通过酶活反应,发现UV-B诱导或暗培养的桑叶中桑辛素氧化环合酶活性增高。利用单因素实验、正交实验对桑辛素氧化环合酶的提取条件进行优化,结果显示提取液pH为7.5,料液比(g/mL)为1:10,提取液浓度为100mM并添加10mM的Na2-EDTA时,浸提时间为2h,反应温度为30℃时,酶比活力最高。上述研究结果为之后桑叶氧化环合酶的分离与纯化奠定了基础。
[Abstract]:Bombyx Mori L. (Bombyx mori.) belongs to Lepidoptera, Silkworm moth family, and Bombyx mori nuclear polyhedrosis virus (BmNPV) disease is the most common and serious type of silkworm disease in silkworm rearing, causing enormous economic losses in silkworm industry. Previous studies showed that mulberry leaves induced by UV-B changed the content of secondary metabolites, and the content of morin N increased significantly. It was found for the first time that mulberry leaves induced by UV-B could improve the resistance of silkworm to BmNPV, and it was speculated that Morin N was the main active component. The differential proteomic analysis of midgut proteins of silkworm by label-free technique revealed the mechanism of inducing mulberry leaves and Morin N resistance. The results provided a new treatment scheme for nuclear polyhedrosis of silkworm and laid a foundation for further study. In addition, the key enzymes inducing the biosynthesis of morin N in mulberry leaves were also studied. The results of this study laid a foundation for studying the changes of secondary metabolites in mulberry leaves induced by UV-B. The main contents and results of this paper are as follows: (1) Photoenzyme-induced changes in mulberry leaves. BmNPV is one of the most harmful viruses to silkworm. In the late stage of BmNPV disease, silkworm often exudes pus and contains a large number of polyhedrosis viruses, commonly known as pyosis. These diseases cause death of silkworm and cause huge economic losses in silkworm industry. The experiment of silkworm anti-virus found that feeding UV-B induced mulberry leaves or coating. After mulberry leaves were fed with mulberry leaves containing Morin N, the resistance of silkworm to BmNPV disease was improved and the mortality of Silkworm Infected with BmNPV was decreased. In order to further study the mechanism of resistance, the midgut of silkworm fed with induced mulberry leaves (BUM) and Morin N (BNM) was analyzed by comparative proteomics. The results showed that 413 and 102 differences were identified between BUM and BNM groups, respectively. Protein. Further analysis by bioinformatics showed that inducing mulberry leaves and Morin N could induce more changes in ribosomal protein expression, regulate protein synthesis and DNA repair, up-regulate ATPase expression and down-regulate ATP synthase expression, thus changing the cellular environment to prevent virus shelling into the cell nucleus and resist virus infection. Morin N can also up-regulate the expression of cytochrome oxidase, Ca2+ related protein and programmed cell death protein, thereby promoting cell apoptosis and inhibiting virus proliferation and replication; up-regulate the expression of lipase-1 and serine protease precursor, thereby enhancing the immunity of silkworm and resisting virus invasion; and up-regulate the expression of histone and Rab proteins. The above results reveal the process of inducing resistance of mulberry leaves and Morin N, which is a new therapeutic scheme for improving the survival rate of Silkworm Infected with BmNPV. (2) Study on the key enzyme of inducing Morin N biosynthesis in mulberry leaves. Morus alba L., mulberry, deciduous tree or shrub, is an important medicinal plant, with a variety of pharmacological effects, but also the main source of silkworm food. Morin is an important secondary metabolite in mulberry leaves, with anti-tumor, anti-inflammatory, anti-virus and other activities. At present, the chemical synthesis of morin N has been reported, but the biosynthesis of morin N has not been reported in detail. The important precursor of biosynthesis of morin N was found to be Morin M. A novel Morin oxidative cyclooxygenase was found in the induced mulberry leaves for the first time. It is important to elucidate the biosynthesis of morin N by catalyzing the cyclization of resveratrol to form Morin M. In addition, the activity of morin oxidative cyclase in mulberry leaves induced by UV-B or cultured in dark was increased by enzyme activity reaction. The extraction conditions of morin oxidative cyclase were optimized by single factor experiment and orthogonal experiment. The results showed that the specific activity of the enzyme was the highest when the pH of the extract was 7.5, the ratio of material to liquid was 1:10, the concentration of the extract was 100 mM and the concentration of Na2-EDTA was 10 mM. The results laid a foundation for the isolation and purification of the oxidative cyclooxygenase from mulberry leaves.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51

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