三角帆蚌贝壳基质蛋白基因hic22和hic74的分离鉴定及其功能探究

发布时间：2018-08-28 13:51

【摘要】：三角帆蚌(Hypriosis cumingii)是我国特有的淡水育珠蚌,所产珍珠占珍珠总产量的80%。已有的研究表明,基质蛋白在贝壳和珍珠的形成过程中发挥着重要作用。有关基质蛋白的研究大多都集中于海水贝类中,在淡水贝类中则相对较少。本实验从三角帆蚌贝壳珍珠层及外套膜组织中克隆得到了2个与珍珠层形成有关的基质蛋白基因,并初步研究了其在珍珠和贝壳形成过程中的作用。1.从三角帆蚌贝壳珍珠层中分离得到了一个分子量大小为22kDa的基质蛋白,根据N端测序结果和已知的基质蛋白基因MSI60的氨基酸序列AAAAAAA分别设计简并引物,从三角帆蚌外套膜中克隆得到了2个基质蛋白基因的cDNA全长序列。其中hic22基因序列全长为731bp,包含一个621bp的开放阅读框,编码206个氨基酸;hic74基因全长2882bp,包含一个2553bp的开放阅读框,编码850个氨基酸,理论分子量为73.7kDa。Hic74的氨基酸组成中富含丙氨酸(30.8%),甘氨酸(25.8%)和丝氨酸(10.6%),在丙氨酸/甘氨酸富集区中,丙氨酸主要以poly A的形式存在,并存在3个酸性区域。组织荧光定量结果显示,hic22和hic74基因在外套膜组织中特异性表达,原位杂交结果显示,hic22和hic74基因主要在外套膜的缘膜部上皮细胞表达,推测基质蛋白hic22和hic74参与了贝壳珍珠层的形成。2.在插片实验中,珍珠颗粒扫描电镜结果显示,珍珠的形成主要分为碳酸钙晶体的无序沉积和有序沉积两个阶段。在规则珍珠层还未形成之前,碳酸钙晶体无序地堆积在表面,而在珍珠层开始形成时,碳酸钙晶体首先在表面成核,然后开始有序的沉积。Hic22和hic74基因在珍珠囊形成和珍珠早期发育时期的荧光定量结果显示,在碳酸钙无序沉积阶段,hic22和hic74基因在珍珠囊中的表达水平极低,而在珍珠成核期表达水平上升,在随后珍珠层的快速生长和平稳生长过程中,两者的表达量呈逐步上升的趋势。说明基质蛋白hic22和hic74在指导碳酸钙成核、调控珍珠层生长过程中发挥着重要作用。3.在破壳修复实验观察中,贝壳先在破碎处形成角质层,而后棱柱层晶体开始在角质层的内表面堆积并快速生长,当棱柱层成熟后,珍珠层晶体开始在棱柱层堆积,且珍珠层的生长方式为典型的“砖墙型”。在贝壳修复的过程中,实验组中hic22基因表达量低于对照组,且呈逐步下降的趋势,推测基质蛋白hic22与其他基质蛋白共同参与贝壳生物矿化过程。而hic74基因则在棱柱层快速生长期和珍珠层快速生长期高表达,推测基质蛋白hic74参与了三角帆蚌贝壳棱柱层和珍珠层的矿化。
[Abstract]:Hyriopsis cumingii (Hypriosis cumingii) is a special freshwater oyster in China, which produces 80% of the pearl. Previous studies have shown that matrix proteins play an important role in the formation of shells and pearls. Most of the studies on matrix proteins are concentrated in seawater shellfish, but relatively few in freshwater shellfish. In this study, two matrix protein genes related to the formation of pearl layer were cloned from the pearl layer and outer membrane tissue of Hyriopsis cumingii, and their roles in the formation of pearl and shell were preliminarily studied. A matrix protein with molecular weight of 22kDa was isolated from the pearl layer of Hyriopsis cumingii shell. Degenerate primers were designed based on N-terminal sequencing results and amino acid sequence AAAAAAA of known matrix protein gene MSI60. The full-length cDNA sequences of two matrix protein genes were cloned from the outer membrane of Hyriopsis cumingii. The total length of hic22 gene is 731 BP, which contains an open reading frame of 621bp, encoding a full length 2882bpof 206 amino acids, and an open reading frame of 2553bp encoding 850 amino acids. The amino acid composition with theoretical molecular weight of 73.7kDa.Hic74 is rich in alanine (30.8%), glycine (25.8%) and serine (10.6%). In the alanine / glycine rich region, alanine mainly exists in the form of poly A, and there are three acidic regions. The results of tissue fluorescence quantitative analysis showed the specific expression of Hech22 and hic74 genes in the outer membrane tissue, and in situ hybridization showed that the genes of Thic22 and hic74 were mainly expressed in the epithelial cells of the marginal membrane of the coat membrane. It is suggested that the matrix proteins hic22 and hic74 are involved in the formation of shell pearl layer. 2. The results of SEM show that the formation of pearls can be divided into two stages: disordered deposition and ordered deposition of calcium carbonate crystals. Before the regular pearl layer was formed, the calcium carbonate crystals piled up disorderly on the surface, and when the pearl layer began to form, the calcium carbonate crystals first nucleated on the surface. Then the fluorescence quantitative results of the sequence deposition of .Hic22 and hic74 genes during the formation of pearl sacs and the early development of pearls showed that the expression levels of Hic22 and hic74 genes in pearl sacs were very low during the disordered deposition of calcium carbonate. However, the expression level of pearls increased during nucleation, and then increased gradually during the rapid and steady growth of pearl layer. The results showed that the matrix proteins hic22 and hic74 played an important role in guiding the nucleation of calcium carbonate and regulating the growth of pearl layer. In the experimental observation of shell breaking and repairing, the shell first formed the cuticle at the broken place, then the prismatic layer crystal began to accumulate on the inner surface of the cuticle and grow rapidly. When the prism layer matured, the pearl layer crystal began to pile up in the prism layer. And the growth mode of pearl layer is typical "brick wall type". In the process of shell repair, the expression of hic22 gene in the experimental group was lower than that in the control group, and gradually decreased. It was speculated that the matrix protein hic22 and other matrix proteins were involved in the process of shell biomineralization. However, hic74 gene was highly expressed during the rapid growth of prisms and pearls, suggesting that the matrix protein hic74 was involved in the mineralization of shell prisms and nacreous layers of Hyriopsis cumingii.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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