本文關鍵詞：珍珠貝貝殼蛋白組學及脅迫環(huán)境下的應激調(diào)控網(wǎng)絡　出處：《中國科學院研究生院(海洋研究所)》2016年博士論文　論文類型：學位論文

  更多相關文章： 合浦珠母貝 蛋白質(zhì)組 生物礦化 WGCNA 環(huán)境脅迫

【摘要】：生物礦化(biomeralization)是指在生物有機大分子(主要是蛋白質(zhì)、糖蛋白或多糖)調(diào)控下,在生物體特定部位有序沉積形成礦物晶體(生物礦物)的過程。軟體動物的貝殼則是生物礦物典型的代表。由于珍珠貝(包括海水和淡水)可分泌形成質(zhì)地堅硬、色彩斑斕、熠熠生輝的文石結晶層,而被利用于培育有核或無核珍珠。合浦珠母貝Pinctada fucata martensii是中國及日本等國家最主要的海水育珠貝,其所產(chǎn)珍珠史稱“南珠”。本研究基于合浦珠母貝全基因組序列全基因組序列精細圖譜,通過檢測和分析貝殼珍珠層與棱柱層的基質(zhì)蛋白(家族),探討貝殼結晶層的形成分子機制;并利用WGCNA的方法構建了貝殼蛋白在非生理條件下的應激網(wǎng)絡。由于珍珠貝珍珠層與珍珠在物質(zhì)組成、形成過程的相似性,該研究對于理解珍珠貝珍珠形成的機制及珍珠品質(zhì)的改善也都有一定的借鑒意義。具體的研究結果如下:1.合浦珠母貝貝殼蛋白組學研究基于合浦珠母貝全基因組序列精細圖譜,本研究利用高效液相色譜串聯(lián)質(zhì)譜電噴霧檢測技術(LC-ESI-MS/MS)分別獲得棱柱層的酸不溶蛋白(PI)、可溶性蛋白(PS)、珍珠層不溶性蛋白(NI)和可溶性蛋白(NS)各207、109、199和120種,其中有78個為已知功能的。分別計算四組蛋白中含量最高的20種蛋白,結果發(fā)現(xiàn)四組樣品共有的高含量基質(zhì)蛋白為酪氨酸酶(Tyrosinase,Tyr)、類纖連蛋白(Fibronectin-like protein,FLP)和N19。棱柱層與珍珠層基質(zhì)蛋白含低復雜性區(qū)域(related low complexity domain,RLCD)蛋白,或稱天然無序蛋白(intrinsically disordered proteins,IDP)。四個樣品中,已報道的珍珠層基質(zhì)蛋白均被檢測到,但含量存在較大的差異,如N19和nacrein等。相同的情況也發(fā)生于已知棱柱層特有的蛋白,如Tyr和FLP。結晶層基質(zhì)蛋白編碼基因的組織表達數(shù)據(jù)顯示,多個組織都存在一定的貢獻率,而非傳統(tǒng)的認識外套膜不同分區(qū)特異表達確定其結晶層特有蛋白。不同的結晶層雖然有其特有的基質(zhì)蛋白,但含量較高的功能基質(zhì)蛋白,一般同時存于兩種結晶層,基質(zhì)蛋白的水平差異可能是選擇不同結晶形態(tài)的主要因素。另外,在基因組數(shù)據(jù)基礎上,比較三種貝類(pinctadafucatamartensii、lottiagigantea和crassostreagigas)的貝殼基質(zhì)蛋白,結果顯示,單拷貝基因家族中的碳酸酐酶(carbonicanhydrase,ca)在貝殼中已經(jīng)分化出新的功能基因(nacrein)。tyr家族有些成員在三個物種中都能找到同源基因,且在合浦珠母貝中為多拷貝基因,而部分成員則僅在長牡蠣中找到同源基因,說明tyr在序列上已經(jīng)發(fā)生明顯分化,這與貝類tyr家族進化分析的結果相吻合。多種ecm在貝殼中含量豐富,暗示ecm在貝殼礦化中起重要的作用。2.貝殼基質(zhì)蛋白(家族)序列分析、同源建模與功能預測對貝殼蛋白中重要的基質(zhì)蛋白(家族)tyr、flp和idps進行了序列和功能分析。分別克隆獲得2個合浦珠母貝和10個長牡蠣tyr家族成員的全長。本研究主要關注合浦珠母貝和長牡蠣貝殼中的tyr成員,其序列特征以及含量分析顯示,tyr的pi值可能對其在貝殼中的功能有重要的相關性。對貝類及其他物種已知的tyr家族進行進化分析,結果顯示合浦珠母貝和長牡蠣tyr都單獨進化成一支,進一步表明兩個物種以及物種內(nèi)功能可能已經(jīng)發(fā)生了分化。同源建模后獲得催化活性中心關鍵氨基酸具體的位置信息,并分析其與酶活性之間的關系。合浦珠母貝的flps序列與結構分析結果顯示,含4個fniii結構域,內(nèi)含氨基酸重復差異較大,二級結構顯示都可形成典型的β折疊,但都缺乏重要的與細胞結合的功能結構域rgd,暗示著與細胞結合的功能的缺少。單個fniii結構域的進化分析顯示,貝殼flps中fniii與高等動物的酪氨酸磷酸酯酶(proteintyrosinephosphatase,ptp)具有較高的同源性,暗示著可能存在相似的功能。合浦珠母貝貝殼中的idps在四個樣品中種類以及豐度占較大的比例。在全基因組水平上,對比3個貝類物種合浦珠母貝、長牡蠣和帽貝貝殼中idps占所有貝殼基質(zhì)蛋白的比例,平均比例分別為28.53%,15.55%和26.64%。發(fā)現(xiàn)在3個物種的特有貝殼基質(zhì)蛋白含量較平均水平高,分別為34.23%、18.52%和34.62%。合浦珠母貝的多拷貝直系同源中含量也達到了33.33%,主要原因為細胞外基質(zhì)(ecm)的組分較多。通過比對發(fā)現(xiàn)珠母貝屬珍珠層和棱柱層IDPs同源性極高,如珍珠層基質(zhì)蛋白MRMP34,NUSP1/2/5/7/8/9和棱柱層PUSP2/8/10。通過網(wǎng)上IDP比對工具,發(fā)現(xiàn)pearlin(N16)、nacrien和MSI60都含有鮑珍珠層特有基質(zhì)蛋白AP7結晶結合位點基序DDNGN。因此推斷文石晶體成核的功能基因通過成核基序(motif)維持其保守性,并以模塊化形式發(fā)揮作用。3.采用共表達網(wǎng)絡分析法(WGCNA)構建合浦珠母貝高溫、酸化脅迫下的應激調(diào)控網(wǎng)絡從NCBI網(wǎng)站CEO數(shù)據(jù)庫中,獲取在高溫和海水酸化脅迫下合浦珠母貝外套膜基因表達數(shù)據(jù)。利用共表達網(wǎng)絡分析法(WGCNA)篩選出在上述脅迫下影響顯著的基因模塊,并對模塊中包含的基因進行功能富集;同時將貝殼蛋白在各個模塊的分布情況進行統(tǒng)計,并分析貝殼蛋白與hub-gene的關聯(lián)性,構建礦化基因的應激網(wǎng)絡。結果表明,高溫顯著響應模塊基因富集結果顯示細胞骨架蛋白基因,如ARHGEF4、SAP和myosin都發(fā)生了顯著的變化,而這些基因主要與細胞遷移關系密切。另外,該模塊中受影響的信號通路基因較多,包含熱激蛋白,主要受影響的基因是膜受體蛋白,說明細胞對溫度應答較復雜。酸化應答模塊共5個,響應的基因包括呼吸相關的血紅素A合成酶、芳香族氨基酸代謝相關的酶、鈣粘蛋白和血小板反應蛋白等。同時,本研究關注的礦化基因也在高溫應答模塊中富集,其中包括Tyr、FLP等重要的礦化基因,說明這些礦化基因?qū)囟容^為敏感。為研究這些礦化基因與其他基因的關聯(lián)性,本研究構建了基因共表達網(wǎng)絡,結果顯示鈣離子結合蛋白、細胞骨架蛋白、蛋白質(zhì)糖基化相關的基因關系密切。在實驗室模擬海洋酸化條件下,測定了3個珍珠層基質(zhì)蛋白編碼功能基因的表達量變化以及貝殼晶體形貌的變化,結果顯示這些基因的表達受不同程度的影響,珍珠層也出現(xiàn)非正常晶體形貌特征。
[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.

【學位授予單位】：中國科學院研究生院(海洋研究所)
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S917.4

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