珍珠贝贝壳蛋白组学及胁迫环境下的应激调控网络

发布时间：2018-01-11 15:37

本文关键词：珍珠贝贝壳蛋白组学及胁迫环境下的应激调控网络　出处：《中国科学院研究生院(海洋研究所)》2016年博士论文　论文类型：学位论文

【摘要】：生物矿化(biomeralization)是指在生物有机大分子(主要是蛋白质、糖蛋白或多糖)调控下,在生物体特定部位有序沉积形成矿物晶体(生物矿物)的过程。软体动物的贝壳则是生物矿物典型的代表。由于珍珠贝(包括海水和淡水)可分泌形成质地坚硬、色彩斑斓、熠熠生辉的文石结晶层,而被利用于培育有核或无核珍珠。合浦珠母贝Pinctada fucata martensii是中国及日本等国家最主要的海水育珠贝,其所产珍珠史称“南珠”。本研究基于合浦珠母贝全基因组序列全基因组序列精细图谱,通过检测和分析贝壳珍珠层与棱柱层的基质蛋白(家族),探讨贝壳结晶层的形成分子机制;并利用WGCNA的方法构建了贝壳蛋白在非生理条件下的应激网络。由于珍珠贝珍珠层与珍珠在物质组成、形成过程的相似性,该研究对于理解珍珠贝珍珠形成的机制及珍珠品质的改善也都有一定的借鉴意义。具体的研究结果如下:1.合浦珠母贝贝壳蛋白组学研究基于合浦珠母贝全基因组序列精细图谱,本研究利用高效液相色谱串联质谱电喷雾检测技术(LC-ESI-MS/MS)分别获得棱柱层的酸不溶蛋白(PI)、可溶性蛋白(PS)、珍珠层不溶性蛋白(NI)和可溶性蛋白(NS)各207、109、199和120种,其中有78个为已知功能的。分别计算四组蛋白中含量最高的20种蛋白,结果发现四组样品共有的高含量基质蛋白为酪氨酸酶(Tyrosinase,Tyr)、类纤连蛋白(Fibronectin-like protein,FLP)和N19。棱柱层与珍珠层基质蛋白含低复杂性区域(related low complexity domain,RLCD)蛋白,或称天然无序蛋白(intrinsically disordered proteins,IDP)。四个样品中,已报道的珍珠层基质蛋白均被检测到,但含量存在较大的差异,如N19和nacrein等。相同的情况也发生于已知棱柱层特有的蛋白,如Tyr和FLP。结晶层基质蛋白编码基因的组织表达数据显示,多个组织都存在一定的贡献率,而非传统的认识外套膜不同分区特异表达确定其结晶层特有蛋白。不同的结晶层虽然有其特有的基质蛋白,但含量较高的功能基质蛋白,一般同时存于两种结晶层,基质蛋白的水平差异可能是选择不同结晶形态的主要因素。另外,在基因组数据基础上,比较三种贝类(pinctadafucatamartensii、lottiagigantea和crassostreagigas)的贝壳基质蛋白,结果显示,单拷贝基因家族中的碳酸酐酶(carbonicanhydrase,ca)在贝壳中已经分化出新的功能基因(nacrein)。tyr家族有些成员在三个物种中都能找到同源基因,且在合浦珠母贝中为多拷贝基因,而部分成员则仅在长牡蛎中找到同源基因,说明tyr在序列上已经发生明显分化,这与贝类tyr家族进化分析的结果相吻合。多种ecm在贝壳中含量丰富,暗示ecm在贝壳矿化中起重要的作用。2.贝壳基质蛋白(家族)序列分析、同源建模与功能预测对贝壳蛋白中重要的基质蛋白(家族)tyr、flp和idps进行了序列和功能分析。分别克隆获得2个合浦珠母贝和10个长牡蛎tyr家族成员的全长。本研究主要关注合浦珠母贝和长牡蛎贝壳中的tyr成员,其序列特征以及含量分析显示,tyr的pi值可能对其在贝壳中的功能有重要的相关性。对贝类及其他物种已知的tyr家族进行进化分析,结果显示合浦珠母贝和长牡蛎tyr都单独进化成一支,进一步表明两个物种以及物种内功能可能已经发生了分化。同源建模后获得催化活性中心关键氨基酸具体的位置信息,并分析其与酶活性之间的关系。合浦珠母贝的flps序列与结构分析结果显示,含4个fniii结构域,内含氨基酸重复差异较大,二级结构显示都可形成典型的β折叠,但都缺乏重要的与细胞结合的功能结构域rgd,暗示着与细胞结合的功能的缺少。单个fniii结构域的进化分析显示,贝壳flps中fniii与高等动物的酪氨酸磷酸酯酶(proteintyrosinephosphatase,ptp)具有较高的同源性,暗示着可能存在相似的功能。合浦珠母贝贝壳中的idps在四个样品中种类以及丰度占较大的比例。在全基因组水平上,对比3个贝类物种合浦珠母贝、长牡蛎和帽贝贝壳中idps占所有贝壳基质蛋白的比例,平均比例分别为28.53%,15.55%和26.64%。发现在3个物种的特有贝壳基质蛋白含量较平均水平高,分别为34.23%、18.52%和34.62%。合浦珠母贝的多拷贝直系同源中含量也达到了33.33%,主要原因为细胞外基质(ecm)的组分较多。通过比对发现珠母贝属珍珠层和棱柱层IDPs同源性极高,如珍珠层基质蛋白MRMP34,NUSP1/2/5/7/8/9和棱柱层PUSP2/8/10。通过网上IDP比对工具,发现pearlin(N16)、nacrien和MSI60都含有鲍珍珠层特有基质蛋白AP7结晶结合位点基序DDNGN。因此推断文石晶体成核的功能基因通过成核基序(motif)维持其保守性,并以模块化形式发挥作用。3.采用共表达网络分析法(WGCNA)构建合浦珠母贝高温、酸化胁迫下的应激调控网络从NCBI网站CEO数据库中,获取在高温和海水酸化胁迫下合浦珠母贝外套膜基因表达数据。利用共表达网络分析法(WGCNA)筛选出在上述胁迫下影响显著的基因模块,并对模块中包含的基因进行功能富集;同时将贝壳蛋白在各个模块的分布情况进行统计,并分析贝壳蛋白与hub-gene的关联性,构建矿化基因的应激网络。结果表明,高温显著响应模块基因富集结果显示细胞骨架蛋白基因,如ARHGEF4、SAP和myosin都发生了显著的变化,而这些基因主要与细胞迁移关系密切。另外,该模块中受影响的信号通路基因较多,包含热激蛋白,主要受影响的基因是膜受体蛋白,说明细胞对温度应答较复杂。酸化应答模块共5个,响应的基因包括呼吸相关的血红素A合成酶、芳香族氨基酸代谢相关的酶、钙粘蛋白和血小板反应蛋白等。同时,本研究关注的矿化基因也在高温应答模块中富集,其中包括Tyr、FLP等重要的矿化基因,说明这些矿化基因对温度较为敏感。为研究这些矿化基因与其他基因的关联性,本研究构建了基因共表达网络,结果显示钙离子结合蛋白、细胞骨架蛋白、蛋白质糖基化相关的基因关系密切。在实验室模拟海洋酸化条件下,测定了3个珍珠层基质蛋白编码功能基因的表达量变化以及贝壳晶体形貌的变化,结果显示这些基因的表达受不同程度的影响,珍珠层也出现非正常晶体形貌特征。
[Abstract]:Biomineralization (biomeralization) refers to the biological macromolecules (mainly protein, glycoprotein or polysaccharide) under the control of the formation of mineral crystals in specific parts of organisms (bio minerals) and orderly deposition process. The software is animal shells biominerals typical representative. The Pearl Oyster (including seawater and freshwater) can secrete the formation of hard texture, colorful, shiny aragonite crystal layer, which is used to develop nuclear or non nuclear. Hepu Pinctada fucata martensii pearl nacre is China and Japan and other countries the main activities for the production of seawater pearl, known as the "pearl pearl". This research is based on the complete genome sequence of the whole genome sequence from Hepu fine mapping, detection and analysis of the nacre and prismatic layer matrix protein (family), to investigate the molecular mechanism of the formation of crystalline shell layer; and using the WGCNA method to construct Shell network stress protein in non physiological conditions. The Pearl Oyster nacre and pearl in material composition, the formation of similar process, the research for understanding the mechanism of the formation of pearl oyster pearl and pearl quality improvement also have certain reference significance. The results are as follows: 1. Hepu pearl shell proteomics full Hepu fine map based on the genome sequence of nacre, using chromatography electrospray tandem mass spectrometry detection technology on the high performance liquid (LC-ESI-MS/MS) were obtained for the prismatic layer of acid insoluble protein (PI), soluble protein (PS), pearl layer of insoluble protein (NI) and soluble protein (NS) in 207109199 and 120. There are 78 known functions were calculated in four groups. In 20 the highest protein protein, the results showed that four samples all have a high content of matrix protein for tyrosinase (Tyrosinase, Tyr), type of fiber Fibronectin (Fibronectin-like protein, FLP) and N19. prismatic layer and pearl layer matrix protein containing a low complexity region (related low complexity domain, RLCD) protein, or natural protein (intrinsically disordered proteins, order IDP). Four samples of nacre matrix proteins have been reported to have been detected, but there is a big differences in content, such as N19 and nacrein. The same situation also occurs in the known prismatic layer specific proteins, such as Tyr and FLP. crystal layer matrix protein encoding gene expression data show that many organizations have a certain contribution rate, rather than the traditional understanding of the mantle in different areas of specific expression to determine the crystal layer the specific proteins of different crystalline layer. Although its unique matrix protein, but the function of matrix protein content high, generally stored in two kinds of crystal layer, differences in the level of matrix protein may choose not to The main factors with the crystal morphology. In addition, the genomic data based on the comparison of three kinds of shellfish (pinctadafucatamartensii, lottiagigantea and crassostreagigas) shell matrix protein, results showed that single copy gene family of carbonic anhydrase (carbonicanhydrase, CA) in the shell has been divided into new functional genes (nacrein).Tyr some members of the family are homologues can be found in the three species, and in Hepu for a multi copy gene from the members, and only in the oyster found homologous genes, indicating that Tyr has obvious differentiation in sequence, and the analysis of shellfish Tyr family evolution results. Many ECM are abundant in the shell, suggesting that ECM plays an important role in the mineralization of.2. shell matrix protein shell (family) in sequence analysis, homology modeling and functional prediction of important protein in shell matrix protein (family) Tyr, FLP and IDPs were analyzed. The full-length sequence and function were cloned from 2 Hepu and 10 oyster Pinctada Tyr family members. The members of the Tyr research mainly focus on Hepu and oyster shell nacre and its sequence characteristics and content analysis showed that the Tyr pI value may have important relevance to the shell of shellfish and other functions. The species known Tyr family by phylogenetic analysis, the results show that the Hepu pearl oyster Tyr separately and evolved into a further two, showed that the species and function may have occurred after differentiation. Homology modeling of location information for the catalytic center of key amino acid specific. And analyze the relationship between enzyme activity and FLPs sequence and structure analysis of Hepu p.martensii shows that containing 4 FNIII domains containing amino acid repeat differences, two level structure The icons are formed typical beta sheet, but with a lack of important cell binding domain RGD, implying lack of binding to cells. The function of a single FNIII domain evolution analysis showed that tyrosine phosphatase FNIII and higher FLPs in animal shells (proteintyrosinephosphatase, PTP) with high homology. Implies that may have similar functions in the shell IDPs. Hepu Pinctada species in four samples and the abundance accounted for a large proportion. At the genomic level, comparison of 3 shellfish species in Hepu IDPs fucata, oyster shells and limpets accounted for all shell matrix protein ratio, average ratio was 28.53% and 15.55%. 26.64%. was found in 3 species of endemic shell matrix protein content higher average, respectively 34.23%, 18.52% and 34.62%. from the Hepu multicopy orthologous content reached 33.33%, the main reason for the extracellular matrix (ECM) components more. Compared with that of Pearl Oyster Pinctada and prismatic layer of IDPs high homology, such as nacre matrix protein MRMP34, NUSP1/2/5/7/8/9 and PUSP2/8/10. IDP prismatic layer through online comparison tools, Pearlin (N16), nacrien and MSI60 have abalone nacre the unique crystal matrix protein AP7 binding site motif DDNGN. therefore infer aragonite crystals into functional gene through nucleation motif (motif) and to maintain its conservative, modular forms play a role by.3. co expression network analysis method (WGCNA) from the construction of Hepu high temperature stress, acid stress regulatory network from the NCBI website in the CEO database, obtained under the stress of Hepu mantle of P. in high temperature and acidification of gene expression data. The co expression network analysis method (WGCNA) was screened in the stress effect The gene module, and module contains the gene function and protein enrichment; shell statistical distribution in each module, and correlation analysis of shell protein and hub-gene gene, construct the mineralization network. The results show that the high temperature stress, significant response module gene enrichment results show cytoskeletal protein genes, such as ARHGEF4. SAP and myosin have undergone significant changes, and these genes are closely related with cell migration. In addition, more affected by signaling genes in the module, including heat shock protein, mainly affected by the gene is that cell membrane receptor protein complex to the temperature response. The acidification response module 5, response the respiratory related genes including heme A synthase, aromatic amino acid metabolism related enzymes, E-cadherin and thrombospondin. At the same time, this research also mineralized gene In the high temperature response module in enrichment, including Tyr, FLP and other important mineralization genes, indicating that these mineralization genes are sensitive to temperature. For the relevance of these mineralized genes and other genes, this study constructed a gene co expression network, results showed that the calcium binding protein, cytoskeletal protein, protein glycosylation related the gene is closely related to ocean acidification conditions. Simulation in the laboratory, the change of the expression of 3 protein encoding genes of nacre matrix and crystal morphology change shells were determined. The results showed that these gene expression was affected by different degree, pearl layer also appeared abnormal crystal morphology.

【学位授予单位】：中国科学院研究生院(海洋研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S917.4

【参考文献】