本文選題：水椰八角鐵甲　切入點(diǎn)：椰扁甲嚙小蜂　出處：《福建農(nóng)林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：寄生蜂在與寄主長期協(xié)同進(jìn)化的過程中建立了一套完整的寄生策略用以突破寄主免疫屏障,這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,毒液被證明能夠調(diào)控寄主的免疫反應(yīng),如黑化反應(yīng)。免疫反應(yīng)響應(yīng)過程中的一些有害中間產(chǎn)物會(huì)對(duì)機(jī)體造成傷害,但絲氨酸蛋白酶抑制劑(serpin)能對(duì)絲氨酸蛋白酶級(jí)聯(lián)反應(yīng)起到負(fù)反饋調(diào)節(jié)作用防止免疫過度。在寄生蜂的毒液組分中存在絲氨酸蛋白酶抑制劑及絲氨酸蛋白酶抑制劑類似物,可以調(diào)節(jié)酚氧化酶原激活因子(prophenoloxidase-activating factor,PPAF)的活性,從而調(diào)控寄主酚氧化酶級(jí)聯(lián)反應(yīng),最終調(diào)節(jié)寄主黑化反應(yīng)。在研究椰扁甲嚙小蜂Tetrastichus brontispae毒液組分的過程中,我們發(fā)現(xiàn)其毒液中存在一個(gè)較高豐度的絲氨酸蛋白酶抑制劑Tbserpin6,因其與寄主水椰八角鐵甲Octodonta nipae中的絲氨酸蛋白酶抑制劑Onserpin6結(jié)構(gòu)相似,推測椰扁甲嚙小蜂毒液蛋白Tbserpin6在調(diào)控寄主免疫反應(yīng)的過程中具有重要作用。為了驗(yàn)證椰扁甲嚙小蜂毒液蛋白Tbserpin6調(diào)節(jié)寄主水椰八角鐵甲酚氧化酶級(jí)聯(lián)反應(yīng)的功能及其可能的調(diào)控機(jī)理,本文克隆了椰扁甲嚙小蜂毒液蛋白Tbserpin6和水椰八角鐵甲Onserpin6、OnPPAF1,并進(jìn)行體外重組表達(dá)驗(yàn)證Tbserpin6、Onserpin6和OnPPAF1在水椰八角鐵甲酚氧化酶激活反應(yīng)中的作用,主要研究結(jié)果如下:(1)克隆獲得Tbserpin6、Onserpin6和OnPPAF1的全長序列分別為969 bp、1248 bp和1164 bp,分別編碼323、416和388個(gè)氨基酸。將OnPPAF1的氨基酸序列與其他昆蟲的酚氧化酶原激活蛋白酶(PPAF、PPAE和PAP)進(jìn)行序列比對(duì)以及系統(tǒng)進(jìn)化分析得知,OnPPAF1為trypsin-like絲氨酸蛋白酶具有典型的N-端"clip"結(jié)構(gòu)域以及C-端絲氨酸蛋白酶結(jié)構(gòu)域,屬CLIPB族成員,具有酰胺酶活性,能激活酚氧化酶酶原。Tbserpin6與Onserpin6同屬于絲氨酸蛋白酶抑制劑家族成員,兩者的反應(yīng)中心環(huán)(RCL)具有較高的相似度,且它們的底物結(jié)合位點(diǎn)P1-P1'都是精氨酸和異亮氨酸,推測Tbserpin6與Onserpin6都能夠調(diào)控水椰八角鐵甲的酚氧化酶原的激活反應(yīng)。(2)利用RNAi技術(shù)驗(yàn)證了 OnPPAF1在水椰八角鐵甲酚氧化酶反應(yīng)中的作用。注射dsOnPPAF1至水椰八角鐵甲蛹體內(nèi)后24h,其血淋巴的酚氧化酶活性及黑化反應(yīng)受到明顯抑制,上述結(jié)果表明OnPPAF1在水椰八角鐵甲的黑化反應(yīng)中起著重要作用。(3)重組蛋白互作結(jié)果表明,Tbserpin6和Onserpin6均能與OnPPAF1互作形成共價(jià)復(fù)合物。重組蛋白Tbserpin6和Onserpin6能夠抑制重組蛋白OnPPAF1的酰胺酶活性,還能抑制水椰八角鐵甲蛹血淋巴酚氧化酶活性與黑化反應(yīng)。綜上,OnPPAF1是水椰八角鐵甲酚氧化酶反應(yīng)中的關(guān)鍵酶,Onserpin6與Tbserpin6皆能同OnPPAF1結(jié)合進(jìn)而對(duì)該酚氧化酶反應(yīng)起到負(fù)反饋調(diào)節(jié)作用。椰扁甲嚙小蜂調(diào)控寄主酚氧化酶級(jí)聯(lián)反應(yīng)的機(jī)理之一可能是該蜂毒液中的Tbserpin6結(jié)構(gòu)與Onserpin6相似,也能與OnPPAF1結(jié)合形成共價(jià)復(fù)合物,進(jìn)而調(diào)控寄主水椰八角鐵甲酚氧化酶級(jí)聯(lián)反應(yīng)。
[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.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S763.306.4

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本文編號(hào)：1560950