椰扁甲啮小蜂毒液蛋白Tbserpin6抑制水椰八角铁甲蛹原酚氧化酶反应的机理

发布时间：2018-03-03 12:02

本文选题：水椰八角铁甲　切入点：椰扁甲啮小蜂　出处：《福建农林大学》2017年硕士论文　论文类型：学位论文

【摘要】：寄生蜂在与寄主长期协同进化的过程中建立了一套完整的寄生策略用以突破寄主免疫屏障,这是寄生蜂成功寄生的第一步。在不含多分DNA病毒(pol ydnavirus,PDV)的寄生蜂中,毒液被证明能够调控寄主的免疫反应,如黑化反应。免疫反应响应过程中的一些有害中间产物会对机体造成伤害,但丝氨酸蛋白酶抑制剂(serpin)能对丝氨酸蛋白酶级联反应起到负反馈调节作用防止免疫过度。在寄生蜂的毒液组分中存在丝氨酸蛋白酶抑制剂及丝氨酸蛋白酶抑制剂类似物,可以调节酚氧化酶原激活因子(prophenoloxidase-activating factor,PPAF)的活性,从而调控寄主酚氧化酶级联反应,最终调节寄主黑化反应。在研究椰扁甲啮小蜂Tetrastichus brontispae毒液组分的过程中,我们发现其毒液中存在一个较高丰度的丝氨酸蛋白酶抑制剂Tbserpin6,因其与寄主水椰八角铁甲Octodonta nipae中的丝氨酸蛋白酶抑制剂Onserpin6结构相似,推测椰扁甲啮小蜂毒液蛋白Tbserpin6在调控寄主免疫反应的过程中具有重要作用。为了验证椰扁甲啮小蜂毒液蛋白Tbserpin6调节寄主水椰八角铁甲酚氧化酶级联反应的功能及其可能的调控机理,本文克隆了椰扁甲啮小蜂毒液蛋白Tbserpin6和水椰八角铁甲Onserpin6、OnPPAF1,并进行体外重组表达验证Tbserpin6、Onserpin6和OnPPAF1在水椰八角铁甲酚氧化酶激活反应中的作用,主要研究结果如下:(1)克隆获得Tbserpin6、Onserpin6和OnPPAF1的全长序列分别为969 bp、1248 bp和1164 bp,分别编码323、416和388个氨基酸。将OnPPAF1的氨基酸序列与其他昆虫的酚氧化酶原激活蛋白酶(PPAF、PPAE和PAP)进行序列比对以及系统进化分析得知,OnPPAF1为trypsin-like丝氨酸蛋白酶具有典型的N-端"clip"结构域以及C-端丝氨酸蛋白酶结构域,属CLIPB族成员,具有酰胺酶活性,能激活酚氧化酶酶原。Tbserpin6与Onserpin6同属于丝氨酸蛋白酶抑制剂家族成员,两者的反应中心环(RCL)具有较高的相似度,且它们的底物结合位点P1-P1'都是精氨酸和异亮氨酸,推测Tbserpin6与Onserpin6都能够调控水椰八角铁甲的酚氧化酶原的激活反应。(2)利用RNAi技术验证了 OnPPAF1在水椰八角铁甲酚氧化酶反应中的作用。注射dsOnPPAF1至水椰八角铁甲蛹体内后24h,其血淋巴的酚氧化酶活性及黑化反应受到明显抑制,上述结果表明OnPPAF1在水椰八角铁甲的黑化反应中起着重要作用。(3)重组蛋白互作结果表明,Tbserpin6和Onserpin6均能与OnPPAF1互作形成共价复合物。重组蛋白Tbserpin6和Onserpin6能够抑制重组蛋白OnPPAF1的酰胺酶活性,还能抑制水椰八角铁甲蛹血淋巴酚氧化酶活性与黑化反应。综上,OnPPAF1是水椰八角铁甲酚氧化酶反应中的关键酶,Onserpin6与Tbserpin6皆能同OnPPAF1结合进而对该酚氧化酶反应起到负反馈调节作用。椰扁甲啮小蜂调控寄主酚氧化酶级联反应的机理之一可能是该蜂毒液中的Tbserpin6结构与Onserpin6相似,也能与OnPPAF1结合形成共价复合物,进而调控寄主水椰八角铁甲酚氧化酶级联反应。
[Abstract]:In the process of host parasitoid and long-term coevolution in the establishment of a complete set of parasitic strategies used to break host immune barrier, this is the first step to success. The parasitic wasps parasitic not containing DNA virus (pol ydnavirus, PDV) of the parasitoid venom, has been proved to be regulation of host immune response. Such as the melanization immune response. In response to some harmful intermediate products in the process of damage to the body, but the serine protease inhibitor (serpin) to the negative feedback regulation to prevent excessive immune to the serine protease cascade reaction. There are serine protease inhibitors and the serine protease inhibitor analogs in the venom group of parasitic wasps, can be adjusted the prophenoloxidase activating factor (prophenoloxidase-activating, factor, PPAF) activity, and regulation of host phenoloxidase cascade regulating host melanization. In the study of Tetrastichus brontispae Tetrastichus brontispae venom components in the process, we found that the Tbserpin6 serine protease inhibitor has a high abundance of its venom, due to the structure of the serine protease inhibitor Onserpin6 and host octodonta nipae Octodonta in nipae is similar to that inferred Tetrastichus brontispae venom protein Tbserpin6 plays an important role in the process of host immune response regulation. In order to verify the Tetrastichus brontispae venom protein Tbserpin6 regulates host octodonta nipae phenoloxidase cascade function and regulation mechanism, we cloned Tetrastichus brontispae venom protein Tbserpin6 and octodonta nipae Onserpin6, OnPPAF1, and the recombinant Tbserpin6 verification Onserpin6 and OnPPAF1 activation in the role of octodonta nipae phenol oxidase, the main results are as follows: (1) cloned the full-length sequence of Onserpin6 Tbserpin6, and OnPPAF1 were 969 BP, 1248 BP and 1164 BP, respectively, encoding 323416 and 388 amino acids. The amino acid sequence of OnPPAF1 and other insect prophenoloxidase activating proteinase (PPAF, PPAE and PAP) sequence and phylogenetic analysis of the ratio was OnPPAF1 trypsin-like is a typical serine protease N- terminal "clip" domain and C- terminal serine protease domain, belonging to the CLIPB family members, has the amidase activity, can activate.Tbserpin6 and Onserpin6 phenol oxidase zymogen members belong to the same family of serine protease inhibitors, the reactive center loop (RCL) has high similarity, and their the substrate binding site P1-P1'is arginine and isoleucine, suggesting that Tbserpin6 and Onserpin6 are both capable of activation of prophenoloxidase regulation of octodonta nipae. (2) use RNAi technology to verify the role of OnPPAF1 in octodonta nipae phenol oxidase reaction. DsOnPPAF1 injection to octodonta nipae pupae after 24h, the activity of phenoloxidase and melanization of hemolymph was significantly inhibited, the results showed that plays an important role in the dark reaction of octodonta nipae OnPPAF1. (3 the recombinant protein interaction) results show that Tbserpin6 and Onserpin6 can interact with OnPPAF1 to form covalent complexes. The amidase activity of recombinant protein Tbserpin6 and Onserpin6 could inhibit the recombinant protein OnPPAF1, can inhibit octodonta nipae hemolymph phenoloxidase activity and melanization. In conclusion, OnPPAF1 is a key enzyme of octodonta nipae phenol oxidase reaction, Onserpin6 and Tbserpin6 can bind OnPPAF1 and the phenol oxidase reaction plays a role of negative feedback regulation. Tetrastichus brontispae regulation of host phenol oxidation The mechanism of enzyme cascade is probably Tbserpin6 structure and Onserpin6 of the venom of similar, can combine with OnPPAF1 to form covalent complexes, and regulation of host octodonta nipae phenoloxidase cascade.

【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S763.306.4

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