IOCAS-IR  > 实验海洋生物学重点实验室
珍珠贝贝壳蛋白组学及胁迫环境下的应激调控网络
Alternative Title珍珠贝
黄荣莲1,2
Subtype博士
Thesis Advisor张国范
2016-05
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline海洋生物学
Keyword合浦珠母贝 蛋白质组 生物矿化 Wgcna 环境胁迫
Other Abstract
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(Pinctada
fucata martensii、  Lottia gigantea 和 Crassostrea gigas)的贝壳基质蛋白,结果显
示,单拷贝基因家族中的碳酸酐酶(carbonic anhydrase,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(Protein 
tyrosine phosphatase, 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。

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Biomineralization refers to an orderly  deposition of mineral crystals (biological 
minerals)  internal  of  the  organism  under  the  control  of  bio-organic  molecules 
(primarily  proteins,  glycoproteins  or  polysaccharides).  Molluscan  shell  is  a  typical 
representative  of  biological  minerals  produced  by  mollusk  species.  Pinctada  fucata 
martensii is a major sea pearl shell in China as well as in Japan. To uphold the quality 
and market value of the pearl, we  detected  and analyzed  of  matrix  protein (or protein 
family)  contained  in  the  prismatic  and  nacreous  layer  of  shell  based  on  fine  pearl 
oyster  genome.  Meanwhile,  we  screen  significantly  response  gene  module(s)  and 
construct  a  biomineralization-related  gene  network  under  non-physiological 
conditions by using WGCNA. Specific contents are as follows:
1. Shell proteomics in Pinctada fucata martensii
By using LC-ESI-MS/MS, we identified acid insoluble ( PI) and soluble proteins 
(PS) in prismatic layer, nacre insoluble (NI) and soluble protein (NS) respectively, as 
207,109,199 and 120 in number . Of the total 366 proteins, 78 are known functional
proteins. The proteins content of top 20 in the 4 samples were calculated. T yrosinase 
(Tyr), fibronectin like protein (FLP) and nacre protein N19 are found in all samples. 
Related low complexity domain (RLCD) proteins, also known as intrinsically 
disordered proteins (IDPs), are high level in nacre and prism layer. The nacre matrix 
proteins such as the N19, nacrein were identified in all four samples, but the levels 
were difference. The same situation also occurs in the prismatic matrix protein, such 
as Tyr, FLP . In combined with the gene expression data, the conclusion of multi-tissue 
contribution to crystal specific gene expression is in contradict with the traditional 
understanding of the mantle regions for different partitions. Finally, we compared
shell matrix proteins of three molluscan species. Novel function gene nacrein derived 
from ancient carbonic anhydrase was detected in all species. Multi-copy genes include Tyr family and ECM coding genes which indicate their important roles in shell 
biomineralization.  
2. Shell protein (family) sequence analysis, homology modeling and functional 
prediction in Pinctada fucata martensii
Clone whole length cDNA 10 Crassostrea gigas Tyrs and 2 P. fucata martensii Tyrs. 
This study focuses on those Tyr family with high abundance in shell which indicate pI 
may be related to its biomineralization function. Phylogenetic analysis indicates 
function differentiation may have occurred. Homology modeling structure of Tyr 
located the key amino acid in the catalytic center, and their relation with the Tyr 
functions and.  Another high level matrix protein family are FLPs with 4 tandem FNIII 
domains. These FNIII domains contain different amino acid repeat but were lack of 
important functional domains RGD found in mammal. Phylogenetic analysis indicate 
the FNIII in FLPs were homologous to those in protein tyrosine phosphatase (PTP) of 
high animals. Sequence and structure analysis FNIII family show the, revealing its 
function conversion. IDPs are abundance in four samples. On the genome level, IDPs 
proportions of total protein shell in three molluscan species were 28.53% in P. fucata 
martensii, 15.55% L. gigantea and 26.64% in C.gigas. Species matrix proteins are 
relative higher proportion of IDPs with 34.23%、18.52%和 34.62% respectively. 
Two types of IDPs were classified by length of the conserved region. One is including 
those that are highly homologous in full length of sequences, such as nacre matrix 
protein MRMP34, NUSP1 /2/5/7/8/9 and the prismatic matrix protein PUSP2 /8/10. 
The other are those contain conserved motifs such as pearlin (N16), nacrien, MSI60, 
which contain crystal nuclear site motif DDNGN of abalone nacre crystalline matrix 
protein AP7. Conservative motifs in nonhomologous aragonite matrix proteins 
represent a common type of modular function.   
3. The network construction for high temperature and ocean acidification stress in 
pearl oyster by co-expression network analysis (WGCNA)  Gene expression data of pearl oyster under high temperatures and seawater
acidification stress from NCBI website CEO database were analyzed by WGCNA. 
One gene module of heat stress and five modules were screened. GO enrichment of 
those modules indicates those functional genes cluster response to environmental 
factor. Cytoskeletal proteins such as ARHGEF4, SAP and myosin were associated to 
temperature stress.  Acidification transponder modules recruit heme A synthase, 
aromatic amino acid metabolism-related enzyme, cadherin and platelet-reactive 
protein. We also focus on signaling pathways in those modules, including the heat 
shock pathway. Among these signaling pathways, membrane receptors are sensitive. 
Interestingly , the high stress module enriched the most mineralization genes, 
including Tyrs and FLPs. To reveal the relation between the mineralization genes and 
other function genes, we constructed the gene co-expression network, showed that 
calcium-binding proteins, cytoskeletal proteins and protein glycosylation genes 
closely related. Simulated the condition of ocean acidification ,we observed changes 
in nacre crystal morphology and examined the expression levels of nacre 
mineralization associated genes (pif177, nacrein and pearlin) in the mantle pallial
zone.
Subject Area海洋生物学 ; 分子生物学
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/117012
Collection实验海洋生物学重点实验室
Affiliation1.中国科学院大学
2.中国科学院海洋研究所
Recommended Citation
GB/T 7714
黄荣莲. 珍珠贝贝壳蛋白组学及胁迫环境下的应激调控网络[D]. 北京. 中国科学院大学,2016.
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