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Molecular Regulation Mechanism of Phenoloxidase-induced Melanin Synthesis in the Mealworm Beetle, Tenebrio Molitor Larvae

论文摘要

由活化的酚氧化酶原所诱导激活的黑化反应能够结合并杀死入侵的外源微生物,是节肢动物先天性免疫反应重要的组成部分。但是如若体内生成过量的醌或者全身黑化反应过度就会对宿主产生毒害,因此黑化反应应有严格的调控体系。但是对于在体内如何调节控制酚氧化酶所诱导黑化反应的分子机制却了解甚少。在这篇论文中通过生化实验证明,在黄粉虫体内Toll途径中能够切割加工酶原Sp（?）tzle的关键酶—Sp（?）tzle加工酶（Tm-SPE）能把分子量为79 kDa的黄粉虫酚氧化酶原（Tm-proPO）切割成分子量分别为76 kDa,46 kDa和27 kDa的片断,可是这些片断都没有产生黑色素的能力。但是如果把Tm-proPO,活性形式的Tm-SPE及黄粉虫发夹样结构域丝氨酸蛋白酶同系物1（TmSPH1）的酶原形式混和在一起进行孵育,则能产生由分子量为76 kDa的Tm-PO和Tm-SPH1的活性形式所组成的高分子量的黑化复合物。而且如果把Tm-SPH1换成Tm-SPH2就不能产生黑化。这种复合物能够在革兰阴性和革兰阳性的细菌表面合成大量的黑化产物因而具有明显的杀菌能力。综上所诉,本研究结果表明在黄粉虫体内Toll途径与酚氧化酶原激活系统这两种主要的先天性免疫反应路径可能是由同一种丝氨酸蛋白酶级联系统所激活的。且在酚氧化酶所诱导的黑化反应中Tm-SPH1作为必需的调节因子能够严格的调控酚氧化原酶联反应的激活以免由于全身过度黑化给宿主带来致命的伤害。

论文目录

  • LIST OF FIGURES
  • ABBREVIATIONS
  • ABSTRACT（IN ENGLISH）
  • ABSTRACT（IN CHINESE）
  • 1.INTRODUCTION
  • 2.MATERIALS AND METHODS
  • 2.1 Materials
  • 2.1.1 Insects and collection of hemolymph
  • 2.1.2 Reagents and buffers
  • 2.2 Measurement of amidase activity
  • 2.3 Determination of melanin synthesis
  • 2.4 Amino acid sequencing
  • 2.5 Antibodies and Western blot
  • 2.6 Purification of Tm-proPO,Tm-SPE and Tm-SPH2 zymogen
  • 2.6.1 Preparation of E1,E2 and E3 fractions by using Toyopearl AF-Heparin HC column
  • 2.6.2 Purification of Tm-pro-PO protein from E2 fraction
  • 2.6.3 Purification of zymogenic and active form of 44 kDa proteins from E2 fraction
  • 2.6.3.1 Purification of zymogenic form of SPE
  • 2.6.3.2 Purification of active form of SPE
  • 2.6.4 Purification of Tm-SPH 1 protein
  • 2.6.5 Purification of Tm-SPH 2 protein from Toyopearl AF-Heparin HC column flow-through fraction
  • 2.7 Injection of protein into Tenebrio Molitor larvae
  • 2.8 Assay of antibacterial activity
  • 3.RESULTS
  • 3.1 The injection of active form of Tm-SPE to Tenebrio larvae induced melanin synthesis in vivo
  • 3.2 Tm-SPE can cleave Tm-proPO into three fractions
  • 3.3 Tm-SPE specifically cleaved Tm-SPH1 but not Tm-SPH2
  • 3.4 Chemically cross-linked adducts are generated by the melanization complex
  • 3.5 Locally concentrated melanin on the surface of bacteria shows bactericidal effect
  • 4.DISCUSSION
  • 5.REFERENCES
  • 6.ACKNOWLEDGEMENT

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