【作者】 杨东；

【导师】 张荣庆；

【作者基本信息】 清华大学 ， 生物学， 2019， 博士

【摘要】 合浦珠母贝(Pinctada fucata)是研究贝类矿化机理的研究物种之一,其珍珠层具有优良的性能而受到关注,对于贝壳形成原理的探索是本领域的研究热点之一。在之前的研究中通过基因芯片测定不同发育时期各基因转录水平的高低,结合基质蛋白特征筛选出若干基质蛋白候选基因,它们的具体功能还需要深入研究。本研究选取其中的一个unigene进行克隆和功能鉴定,命名为mantle protein N25(简称N25),论文首先深入分析了N25蛋白的功能和影响矿化的机理。通过RACE克隆获得了N25基因的全长,检测到该基因在外套膜组织中特异性表达。本研究使用原核表达纯化难溶性基质蛋白N25的包涵体,再利用蛋白质重折叠获得了具有活性的可溶性蛋白,初步建立一种通过包涵体复性获得难溶性基质蛋白的方案。纯化获得的N25蛋白具有结合方解石、文石和几丁质的能力。贝壳组分的免疫印迹显示N25蛋白分布于棱柱层和珍珠层的不可溶性组分。N25蛋白对方解石和文石的结晶形貌都有明显的影响,我们使用结构光照明显微镜对Cy5标记的N25在方解石上的分布进行了成像定位,结果显示N25蛋白只分布在晶体表面而不进入晶体内部。借助软件计算模拟的方法,我们得到了符合预期的方解石形貌模拟,并推测N25蛋白通过结合晶体特定晶面和降低晶面吸附能的方式调控方解石外形。此外,我们通过真核细胞表达N25-EGFP蛋白的方法跟踪观察N25蛋白的分泌过程,研究表明N25通过细胞囊泡分泌的方式离开细胞。通过使用SDS、DTT分步处理贝壳珍珠层和棱柱层的不溶性框架,我们从中洗脱下来了不同的基质蛋白组分。N19蛋白富含较多的Cys氨基酸,在DTT处理的时候从框架上解离下来,推测N19可能通过二硫键连接在其他大分子上。一些框架蛋白以几丁质结合结构域与框架产生非共价的连接,在SDS处理后较容易解离下来。而一些基质蛋白既没有Cys残基也没有几丁质结合域,却能够牢靠地结合框架。这些现象表明,贝壳框架的组成和组装是多层次而且复杂的。更多还原

【Abstract】 Pinctada fucata is one of the species to study the mineralization mechanism of shellfish since its nacreous layer has excellent performance.The principle of shell formation is one of the research hotspots in this field.In the previous study,the transcription level of each gene in different developmental stages was determined by gene chip,and several matrix protein candidate genes were screened according to the characteristics of matrix proteins,however,their functions need further identification.In this research,one of these unigenes was functionally identified and we firstly analyzed the effects of mantle protein N25(N25)on mineralization.The full length of N25 was obtained by RACE,which was specifically expressed in the mantle tissue.After purifying the inclusion bodies of N25,the active soluble protein was obtained by protein refolding.A scheme for obtaining poorly soluble matrix proteins by renaturation of inclusion bodies was preliminarily established.The purified N25 has the ability to bind calcite,aragonite,and chitin.Immunoblotting of the shell fraction revealed N25 was distributed in the insoluble component of both the nacre and prism layer.N25 could alter the morphology of both calcite and aragonite.We used a SIM microscope to image the distribution of cy5-labeled N25 on calcite.The results show that N25 protein is only distributed on the crystal surface.With the help of the simulation method,we obtained the predicted calcite morphology.We speculated that N25 protein regulates the calcite shape by binding to the specific crystal planes and reducing the attachment energy.We traced the secretion of N25 by eukaryotic expression of N25-EGFP protein and observed the secretion by the vesicle.By using SDS and DTT to extract the insoluble framework of the nacre and the prism layer,we eluted different matrix protein components.The N19 protein is rich in Cys and is dissociated from the framework during DTT treatment.It is speculated that N19 may be linked to other macromolecules via disulfide bonds.Some framework proteins produce a non-covalent linkage to the framework with a chitin-binding domain that is easier to dissociate after SDS treatment.Some matrix proteins have neither the Cys nor the chitin-binding domain but they could still bind to the framework firmly.These phenomena indicate that the composition and assembly of the shell frame are multi-layered and complex.更多还原