间液蛋白在合浦珠母贝贝壳形成中的作用机制研究

发布时间：2018-01-05 13:33

本文关键词：间液蛋白在合浦珠母贝贝壳形成中的作用机制研究　出处：《清华大学》2016年博士论文　论文类型：学位论文

【摘要】：合浦珠母贝(Pinctada fucata)贝壳由珍珠层和棱柱层两种矿化层组成,其形成过程涉及一个矿化平衡的过程。间液位于珠母贝贝壳和外套膜之间,被认为在贝壳形成中起重要作用。然而,对间液的功能研究却相对较少。碳酸钙体外结晶实验表明间液蛋白不仅能够控制CaCO_3晶体的形貌,而且能够通过文石和方解石特异结合蛋白调控CaCO_3的晶型转变。结晶抑制实验表明,当CaCl2和NaHCO_3的浓度从50mM降低到5mM时,间液蛋白从显著增加CaCO_3晶体的数量转变为抑制CaCO_3的沉积。间液蛋白同时能够显著抑制CaCO_3结晶速率。在体内,当间液被连续抽取20天后,间液蛋白浓度降低,珍珠层不能形成正常的文石小片,取而代之的是无序沉积的方解石。同时,间液变化能够作为外部信号引起珍珠层和棱柱层形成相关基因的变化。综上所述,间液蛋白不但参与CaCO_3结晶的成核,形貌,抑制和相变,而且在贝壳形成特别是珍珠层棱柱层转换中起着双重作用。随后我们对特异结合方解石和文石的间液蛋白进行了LC-MS质谱鉴定,共鉴定到了221条共有序列,79条方解石特异结合的序列和47条文石特异结合的序列。氨基酸组成分析发现与方解石特异结合的蛋白其Lys,Val,Ser和Gly含量相对较多,而与文石特异结合的蛋白其Leu,Glu和Val相对较多。结构域分析发现与方解石文石特异结合的蛋白结构域涉及离子调节,蛋白相互作用,蛋白酶抑制和胶原蛋白等,这些结果为后续研究提供了基础。最后,我们发现间液蛋白SPARC的含量在贝壳缺刻后48 h显著升高。SPARC在贝壳珍珠层及棱柱层EDTA可溶性组分中均存在。当用多克隆抗体将间液中SPARC拮抗掉以后,新生成珍珠小片受到明显影响。在体外,SPARC不但能够控制CaCO_3晶体的形貌而且能在方解石结晶体系中诱导球文石的形成。然而,Mg2+能够抵消这种作用,并诱导文石的形成。进一步的圆二色谱实验表明,SPARC可能通过其蛋白二级结构的改变来发挥作用。结合结构域分析,我们发现其EC结构域在稳定球文石中起主要作用。综上所述,SPARC通过其EC结构域稳定球文石,从而抑制方解石的形成,以便在Mg2+的作用下促进文石的形成来参与贝壳的形成。综上所述,我们第一次深入研究了间液蛋白在珠母贝贝壳矿化平衡中的双重作用,并且发现间液中的蛋白能够控制ACC,球文石,方解石和文石之间的转变。我们的研究结果为珍珠生产和体外类似材料的合成提供了有力的理论指导。
[Abstract]:Hepu p.margaritifera (Pinctada fucata) by pearl shell and prismatic layer two mineralization layer, its forming process involves a balance between mineralization process. The liquid is located between the shell and the nacre mantle, is thought to play an important role in the formation of shell. However, the liquid between the function of carbonate is relatively small. In vitro experiments showed that the morphology of crystalline calcium liquid between protein can not only control the CaCO_3 crystal, but also the specific binding of aragonite and calcite crystal transition regulatory protein CaCO_3. Crystallization inhibition experiments showed that when the concentration of CaCl2 and NaHCO_3 decreased from 50mM to 5mM, significantly increasing the number of interstitial fluid proteins from the transition of CaCO_3 crystal for inhibiting deposition CaCO_3. At the same time between liquid protein can significantly inhibit the crystallization rate of CaCO_3. In vivo, when the liquid is extracted continuously between the 20 days between liquid protein concentration decreased, pearl layer cannot form normal aragonite Scrap, instead of the disordered deposition of calcite. At the same time, between the liquid can be used as an external signal caused by the change of Pearl and prismatic layer formation and changes of related genes. In summary, nuclear, a liquid protein involved in not only the CaCO_3 crystallization morphology, inhibition and transformation, but also in the shell formation especially plays a dual role in the nacre prism layer conversion. The specific binding of calcite and aragonite interstitial fluid proteins were identified by LC-MS mass spectrometry, identified 221 common sequence, sequence specific binding of the calcite combined with 79 sequences and 47 specific provisions of stone. The amino acid composition analysis showed that combining with calcite specific protein Lys, Val, Ser and the content of Gly is relatively more, and combined with the aragonite specific protein Leu, Glu and Val are relatively more. Domain analysis showed that the protein binding domains and specific ion calcite aragonite to adjust, Protein interaction, protease inhibitor and collagen. These results provide a basis for further study. Finally, we found that the content of liquid protein SPARC in the shell after 48 h significantly increased.SPARC notch exist in nacre and prismatic EDTA soluble fraction. When SPARC with polyclonal antibody will be between liquid the new generation of small pearls off the antagonist, significantly affected. In vitro, SPARC can not only control the formation of the morphology of CaCO_3 crystal and can induce vaterite in calcite crystallization system. However, Mg2+ can counteract the effect, and the formation of aragonite. That induced by round two chromatography experiments further, SPARC may play a role in the two stage structure. The change of the protein binding domain analysis, we found that the EC domain plays a major role in the stability of vaterite. To sum up, SPARC through its EC domain of Qiu Wen Stone, thus inhibiting the formation of calcite, aragonite formation in order to promote the formation of the shell to participate in the presence of Mg2+. In conclusion, we first investigate the dual role of interstitial fluid proteins in nacre shell mineralization in the balance, and found that the liquid between the protein can control the ACC, transition between vaterite, calcite and aragonite. Our results provide strong theoretical guidance for the synthesis of pearl production in vitro and similar materials.

【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q51

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