基于多组学分析的马氏珠母贝矿化相关基因研究
发布时间：2018-08-15 18:41
【摘要】：软体动物贝壳是典型的生物矿化产物。马氏珠母贝Pinctada fucata martensii是研究生物矿化的良好材料。本研究以课题组组装获得的马氏珠母贝基因组精细图为参考,通过比较基因组、蛋白质组及转录组等多组学数据关联分析,研究参与贝壳形成的基质蛋白家族及其进化过程、发现与之相关的代谢通路和调控因子;利用RNA干扰、酵母双杂交以及双荧光素酶报告系统等多种研究方法对PmRunt上游信号分子、下游效应基因及其转录后调控因子的作用机制进行了研究。研究结果如下:1、组学分析发现矿化相关基因家族及调控因子(1)马氏珠母贝基因组进化分析本研究对马氏珠母贝、长牡蛎、帽贝、海蜗牛、章鱼、小头虫、水蛭、斑马鱼和人九个物种基因组序列进行基因家族聚类并对扩张和收缩的基因家族进行分析。以软体动物有壳类四个物种共同祖先与无壳的头足类章鱼的比较,发现贝壳形成的共同机制,即钙离子信号通路及矿质离子吸收、糖胺聚糖和多聚氨基糖生物合成及代谢为贝壳的形成提供了物种基础。对马氏珠母贝扩张收缩的基因家族进行分析,发现磺基转移酶扩张与贝壳珍珠层中高丰度的酸性粘多糖的性状相一致。对马氏珠母贝和长牡蛎共同扩张的贝壳蛋白家族进行分析,发现类纤连蛋白(FNL)可能在二者贝壳形成中均发挥重要作用;酪氨酸酶家族(TYR)的扩张可能导致马氏珠母贝贝壳酪氨酸酶蛋白独立分支的出现;含有VWA domain的蛋白家族(VWAP)可能对马氏珠母贝珍珠层形成的贡献力更大。对马氏珠母贝特有基因家族进行分析,发现具有氨基酸偏好性和重复单元的特有贝壳蛋白家族RLCD(related low complexity domain)在马氏珠母贝贝壳形成中发挥重要作用。(2)马氏珠母贝发育及组织转录组分析本研究以马氏珠母贝基因组精细图为参考,对马氏珠母贝发育及组织转录组样品进行转录组测序和分析。(1)担轮前期到担轮期、眼点期到变态后时期贝壳蛋白表达明显上调,分别与贝壳原壳和次生壳的形成一致,提示这两个阶段是贝壳形成的重要时期。(2)除外套膜组织是贝壳形成的主要器官,血细胞和闭壳肌对矿化基因也具有一定贡献。(3)对基因组进化分析获得的马氏珠母贝扩张的和特有的矿化相关基因家族表达模式进行分析,发现几丁质合成和代谢相关酶、磺基转移酶、TYRs、VWAPs、FNLs以及RLCDs,均有部分基因在担轮期或变态后时期表达量上调,同时在外套膜组织或珍珠囊中高表达,提示基因家族的倍增和基因功能分化、以及矿化相关种系特有基因的出现可能在贝壳的形态进化及形成中发挥重要作用。(4)分析了已知矿化相关调控因子的表达模式,同时对两个关键时期转录组差异表达基因进行功能富集,发现Wnt、BMPs、VEGF、MAPK、破骨细胞分化等信号通路以及Runt、SMAD、AP-1等转录因子可能在贝壳形成的调控中发挥重要作用;并且雌激素、维生素D3、甲状腺素和蜕皮激素可能参与变态后次生壳的形成。2、转录因子PmRunt及相关通路调控珍珠层的形成(1)PmRunt对珍珠层形成的调控通过组学的分析筛选获得可能参与贝壳形成的重要转录因子PmRunt,利用RACE技术对PmRunt进行了克隆,并对其配体PmCBF的CDS区进行了验证。PmRunt基因全长2319bp,5`端非编码区为82bp,3`端非编码区为599bp,编码的蛋白具有545个氨基酸。PmRunt含有典型的具有DNA结合能力的Runt结构域(RHD),-COOH端存在VWRPY modif。PmRunt与脊椎动物的RUNX1相似度最高,系统进化分析发现脊椎动物和无脊椎动物的Runt protein分为独立的两支,软体动物的Runt protein来自同一祖先。酵母双杂交结果证明PmRunt和PmCBP蛋白之间存在相互作用。为了研究PmRunt对贝壳形成的影响,本研究以注射dsRFP组为阴性对照、利用dsRNA介导的RNA干扰使得PmRunt的表达发生显著性下调(P0.05),扫描电镜检测贝壳内表面,发现珍珠层的形成发生紊乱,暗示PmRunt参与贝壳珍珠层的形成。转录组测序发现多个珍珠层基质蛋白基因表达量在PmRunt干扰组发生显著性下调,其中包括carbonic anhydrase-like、TYR、VWAP、Mantle gene4(MG4)等。利用RO5-3335抑制剂抑制PmRunt与PmCBF的结合后,MG4(Pma_318.183和Pma_10000069)、VWAP(Pma_10019836)、TYR2(Pma_10029257)、Nacrein的表达量均发生显著性下调(P0.05),暗示这些基因可能受到PmRunt的直接调控。因此,本研究利用双荧光素酶报告系统检测了PmRunt与VWAP(Pma_10019836)和Nacrein启动子区域的相互作用,发现PmRunt可以增强VWAP和Nacrein启动子的转录活性,提示PmRunt可能直接参与VWAP和Nacrein基因表达的调控。(2)PmRunt调控的MG4及其同源基因(C1qDC)参与贝壳珍珠层的形成MG4作为PmRunt下游的效应基因,属于C1q domain containing protein(C1qDC)家族。本研究对马氏珠母贝和长牡蛎发生扩张的C1qDCs的表达模式进行了分析,并对PmRunt调控的C1qDCs(Pma_318.183、Pma_576.501、Pma_10000069、Pma_10012374)在贝壳矿化中的功能进行研究。结果显示,尽管两个物种中C1qDC均出现明显的扩张现象,但马氏珠母贝中具有外套膜高丰度表达的集合,提示扩张的C1qDC参与贝壳矿化。利用RNA干扰技术显著性下调Pma_318.183(MG4)、Pma_576.501和Pma_10012374在外套膜套膜区的表达(P0.05),SEM检测发现3个实验组的贝壳内表面珍珠层的形成发生了紊乱,因此C1qDCs可能影响贝壳珍珠层的形成。(3)蜕皮激素对PmRunt及贝壳矿化相关基因表达的调控在发育转录组比较分析中,发现蜕皮激素可能参与贝壳的形成。本研究对蜕皮激素受体及其结合蛋白RXR的CDS序列进行了验证。通过Elisa方法检测贝壳损伤后血清中蜕皮激素含量的变化,发现蜕皮激素在缺壳后2h显著性上调(P0.05),4h时的浓度最高,而36h的上调与贝壳损伤修复膜的出现一致,暗示蜕皮激素可能与贝壳的修复相关。利用40ng/L的蜕皮激素浸泡处理外套膜套膜区小片,转录因子PmRunt、AP-1、BMP2/7、VWAP、TYR2、KRMP和CHS的表达均发生显著性变化(P0.05),以上结果提示蜕皮激素可能调控PmRunt的表达,同时也参与其他矿化相关基因的调控影响贝壳的形成。3、microRNA调控PmRunt及NF-κB通路基因表达本研究利用体外细胞实验和Pm-miR-183活体过表达实验检测Pm-miR-183对PmRunt的调控作用,结果显示Pm-miR-183与包含PmRunt的3`UTR区域的pmiRreport载体共转染293T细胞后,荧光素酶活性发生显著性下调(P0.05);注射PmmiR-183 mimics类似物后,马氏珠母贝外套膜组织中的Pm-miR-183表达量发生显著性上调(P0.05),PmRunt的表达量发生显著性下调(P0.05),同时珍珠层的形成发生紊乱,以上结果表明Pm-miR-183可能通过与PmRunt的3`-UTR互作抑制基因的表达,参与珍珠层形成的调控。本研究利用生物信息学分析获得马氏珠母贝Pm-miR-146a,并利用茎环引物特异性反转录,qRT-PCR检测Pm-miR-146a在不同组织的表达量,结果显示Pm-miR-146a在各个组织中均有表达,在外套膜组织的表达量较高,在血液中的表达量较低。miRanda软件预测发现Pm-miR-146a与巨噬细胞迁移抑制因子(MIF)存在靶向作用,注射Pm-miR-146a mimics类似物后,血细胞中Pm-miR-146a的表达量发生显著性上调,同时MIF和NF-κB的表达量发生显著性下调(P0.05)。以上结果暗示Pm-mi R-146a可能通过调控MIF的表达参与NF-κB通路的负调控。
[Abstract]:Mollusk shells are typical biomineralization products. Pinctada fucata martensii is a good material for studying biomineralization. This study is based on the genome fine map of Pinctada martensii assembled by our research group. Through comparative genome, proteomic and transcriptome data association analysis, we participated in the study of mollusk shells. The matrix protein family and its evolution process were found to be related to the metabolic pathways and regulatory factors. The mechanism of PmRunt upstream signaling molecules, downstream effector genes and post transcriptional regulatory factors was studied by means of RNA interference, yeast two hybrid and dual luciferase reporter system. The results are as follows: 1. Genomic analysis revealed that mineralization-related gene families and regulatory factors (1) Genomic evolution of Pinctada martensii. Comparing the common ancestors of four species of molluscs with those of the shell-less cephalopod octopus, we found that the common mechanism of shell formation, i.e. calcium signaling pathway and mineral ion absorption, glycosaminoglycan and polyaminoglycan biosynthesis and metabolism, provided a species basis for shell formation. Geneticists of expansion and contraction of Pinctada martensii Sulfotransferase dilatation was found to be consistent with the high abundance of acid mucopolysaccharides in the nacre of the shellfish. The analysis of the shell protein family co-expanded by Pinctada martensii and Oyster gigas revealed that fibronectin-like protein (FNL) may play an important role in shell formation of both species; the expansion of tyrosinase family (TYR) may be involved. The protein family containing VWA domain (VWAP) may contribute more to the formation of Pearl layers in Plutella martensii. The analysis of the specific gene family of Plutella martensii revealed that the RLCD (related shell protein family) with amino acid preference and repetitive units was a unique shell protein family. Low complexity domain plays an important role in the formation of pearl oyster shells. (2) The development and transcriptome analysis of Pinctada martensii were carried out by sequencing and analyzing the development and tissue transcriptome samples of Pinctada martensii based on the genome fine map. (1) From the early stage of the stretcher to the stretcher stage, the eyespot stage changed. The expression of shell protein was significantly up-regulated during the postnatal period, which was consistent with the formation of shell protoshell and secondary shell, suggesting that the two stages were the important stages of shell formation. (2) Apart from mantle tissue, the main organs of shell formation, blood cells and adductor muscles also contributed to mineralization genes. (3) Pearl martensii obtained from genome evolution analysis. Expansion and specific expression patterns of mineralization-related genes family in female shellfish were analyzed. Chitin synthesis and metabolism-related enzymes, sulfotransferase, TYRs, VWAPs, FNLs and RLCDs were found to be up-regulated in some genes at the stage of carousel or post-metamorphosis, and high in mantle tissues or pearl sacs, suggesting that the gene family was doubled. (4) The expression patterns of known mineralization-related regulatory factors were analyzed, and the differentially expressed genes in the transcriptomes of the two critical stages were enriched. Wnt, BMPs, VEGF, MAPK, osteoclasts were found. Chemical signaling pathways and transcription factors such as Runt, SMAD, AP-1 may play an important role in the regulation of shell formation, and estrogen, vitamin D3, thyroid hormone and ecdysone may participate in the formation of metamorphic secondary shell. 2, transcription factor PmRunt and related pathways regulate the formation of nacre (1) PmRunt regulates the formation of nacre. PmRunt was cloned by RACE and its ligand PmCBF CDS region was validated. The PmRunt gene was 2319 BP in length, 82 BP in the 5 `non-coding region, 599 BP in the 3 `non-coding region. The encoded protein contained 545 amino acids. Phylogenetic analysis showed that the Runt proteins of vertebrates and invertebrates were divided into two separate branches, and the Runt proteins of molluscs came from the same ancestor. The results of yeast two-hybrid showed that PmRunt and PmCBP eggs were similar. In order to study the effect of PmRunt on the formation of shells, the expression of PmRunt was significantly down-regulated by RNA interference mediated by dsRNA (P 0.05). Scanning electron microscopy showed that the formation of nacre was disordered, suggesting that PmRunt was involved in the formation of nacre. Transcriptional sequencing revealed that the expression of several nacre matrix protein genes was significantly down-regulated in the PmRunt interference group, including carbonic anhydrase-like, TYR, VWAP, and Mantle gene 4 (MG4). Inhibiting the binding of PmRunt to PmCBF by RO5-3335 inhibitors, MG4 (Pma_318.183 and PMA_10000069), VWAP (Pma_10019836), TYR2 (Pma_1002925) were detected. 7) The expression of Nacrein was significantly down-regulated (P 0.05), suggesting that these genes may be directly regulated by PmRunt. Therefore, the interaction between PmRunt and VWAP (Pma_10019836) and Nacrein promoter regions was detected by using a double luciferase reporter system. PmRunt could enhance the transcriptional activity of VWAP and Nacrein promoters. These results suggest that PmRunt may be directly involved in the regulation of VWAP and Nacrein gene expression. (2) MG4 and its homologous gene (C1qDC) regulated by PmRunt are involved in the formation of nacre in shells. MG4, as an effector gene downstream of PmRunt, belongs to the C1q domain containing protein (C1qDC) family. The function of PmRunt-regulated C1qDCs (Pma_318.183, Pma_576.501, Pma_10000069, Pma_10012374) in shell mineralization was studied. The results showed that although C1qDCs in both species exhibited evident dilatation, there was a high abundance of mantle expression in Pinctada martensii, suggesting that expanded C1qDCs participated in shell mineralization. The expression of Pma_318.183 (MG4), Pma_576.501 and PMA_10012374 in mantle envelope was significantly down-regulated by RNA interference technique (P 0.05). The formation of nacre on the inner surface of shells in three experimental groups was disordered by SEM. Therefore, C1qDCs may affect the formation of nacre in shells. (3) Ecdysterone may affect the formation of PmRunt and mineralized shells. In this study, the CDS sequence of ecdysone receptor and its binding protein RXR was validated. Elisa method was used to detect the changes of ecdysone content in serum after shell injury, and it was found that ecdysone was significantly involved in shell formation 2 hours after shell injury. Upregulation (P 0.05), the highest concentration at 4 h, and 36 h up-regulation was consistent with the appearance of shell repair membrane, suggesting that ecdysone may be related to shell repair. These results suggest that ecdysone may regulate the expression of PmRunt and other mineralization-related genes. 3. MicroRNA regulates the expression of PmRunt and NF-kappa B pathway genes. In this study, Pm-microRNAs were used to detect the regulation of Pm-microRNAs on PmRunt in vitro and in vivo overexpression of Pm-microRNAs-183. Luciferase activity was significantly down-regulated (P 0.05) after R-183 was co-transfected into 293T cells with pMireport vector containing 3 `UTR region of PmRunt; Pm-microRNA-183 expression was significantly up-regulated (P 0.05) and PmRunt expression was significantly down-regulated (P 0.05) after Pm miR-183 MICs analogue was injected into Mantle Tissues of Pinctada martensii. These results suggest that Pm-microRNA-183 may be involved in the regulation of nacre formation by interacting with PmRunt's 3'-UTR suppressor gene expression. Pm-microRNA-146a was obtained from Pinctada martensii by bioinformatics analysis, and the expression of Pm-microRNA-146a in different tissues was detected by stem-loop primer specific reverse transcription and qRT-PCR. The results showed that Pm-microRNA-146a was expressed in all tissues, higher in mantle tissues, and lower in blood. MicroRNAanda software predicted that Pm-microRNA146a and macrophage migration inhibitor (MIF) had a targeting effect. After injection of Pm-microRNA146a MICs analogue, the expression of Pm-microRNA146a in blood cells was increased. These results suggest that Pm-mi R-146 a may participate in the negative regulation of NF-kappa B pathway by regulating the expression of MIF.
【学位授予单位】：广东海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4
，
本文编号：2185072
[Abstract]:Mollusk shells are typical biomineralization products. Pinctada fucata martensii is a good material for studying biomineralization. This study is based on the genome fine map of Pinctada martensii assembled by our research group. Through comparative genome, proteomic and transcriptome data association analysis, we participated in the study of mollusk shells. The matrix protein family and its evolution process were found to be related to the metabolic pathways and regulatory factors. The mechanism of PmRunt upstream signaling molecules, downstream effector genes and post transcriptional regulatory factors was studied by means of RNA interference, yeast two hybrid and dual luciferase reporter system. The results are as follows: 1. Genomic analysis revealed that mineralization-related gene families and regulatory factors (1) Genomic evolution of Pinctada martensii. Comparing the common ancestors of four species of molluscs with those of the shell-less cephalopod octopus, we found that the common mechanism of shell formation, i.e. calcium signaling pathway and mineral ion absorption, glycosaminoglycan and polyaminoglycan biosynthesis and metabolism, provided a species basis for shell formation. Geneticists of expansion and contraction of Pinctada martensii Sulfotransferase dilatation was found to be consistent with the high abundance of acid mucopolysaccharides in the nacre of the shellfish. The analysis of the shell protein family co-expanded by Pinctada martensii and Oyster gigas revealed that fibronectin-like protein (FNL) may play an important role in shell formation of both species; the expansion of tyrosinase family (TYR) may be involved. The protein family containing VWA domain (VWAP) may contribute more to the formation of Pearl layers in Plutella martensii. The analysis of the specific gene family of Plutella martensii revealed that the RLCD (related shell protein family) with amino acid preference and repetitive units was a unique shell protein family. Low complexity domain plays an important role in the formation of pearl oyster shells. (2) The development and transcriptome analysis of Pinctada martensii were carried out by sequencing and analyzing the development and tissue transcriptome samples of Pinctada martensii based on the genome fine map. (1) From the early stage of the stretcher to the stretcher stage, the eyespot stage changed. The expression of shell protein was significantly up-regulated during the postnatal period, which was consistent with the formation of shell protoshell and secondary shell, suggesting that the two stages were the important stages of shell formation. (2) Apart from mantle tissue, the main organs of shell formation, blood cells and adductor muscles also contributed to mineralization genes. (3) Pearl martensii obtained from genome evolution analysis. Expansion and specific expression patterns of mineralization-related genes family in female shellfish were analyzed. Chitin synthesis and metabolism-related enzymes, sulfotransferase, TYRs, VWAPs, FNLs and RLCDs were found to be up-regulated in some genes at the stage of carousel or post-metamorphosis, and high in mantle tissues or pearl sacs, suggesting that the gene family was doubled. (4) The expression patterns of known mineralization-related regulatory factors were analyzed, and the differentially expressed genes in the transcriptomes of the two critical stages were enriched. Wnt, BMPs, VEGF, MAPK, osteoclasts were found. Chemical signaling pathways and transcription factors such as Runt, SMAD, AP-1 may play an important role in the regulation of shell formation, and estrogen, vitamin D3, thyroid hormone and ecdysone may participate in the formation of metamorphic secondary shell. 2, transcription factor PmRunt and related pathways regulate the formation of nacre (1) PmRunt regulates the formation of nacre. PmRunt was cloned by RACE and its ligand PmCBF CDS region was validated. The PmRunt gene was 2319 BP in length, 82 BP in the 5 `non-coding region, 599 BP in the 3 `non-coding region. The encoded protein contained 545 amino acids. Phylogenetic analysis showed that the Runt proteins of vertebrates and invertebrates were divided into two separate branches, and the Runt proteins of molluscs came from the same ancestor. The results of yeast two-hybrid showed that PmRunt and PmCBP eggs were similar. In order to study the effect of PmRunt on the formation of shells, the expression of PmRunt was significantly down-regulated by RNA interference mediated by dsRNA (P 0.05). Scanning electron microscopy showed that the formation of nacre was disordered, suggesting that PmRunt was involved in the formation of nacre. Transcriptional sequencing revealed that the expression of several nacre matrix protein genes was significantly down-regulated in the PmRunt interference group, including carbonic anhydrase-like, TYR, VWAP, and Mantle gene 4 (MG4). Inhibiting the binding of PmRunt to PmCBF by RO5-3335 inhibitors, MG4 (Pma_318.183 and PMA_10000069), VWAP (Pma_10019836), TYR2 (Pma_1002925) were detected. 7) The expression of Nacrein was significantly down-regulated (P 0.05), suggesting that these genes may be directly regulated by PmRunt. Therefore, the interaction between PmRunt and VWAP (Pma_10019836) and Nacrein promoter regions was detected by using a double luciferase reporter system. PmRunt could enhance the transcriptional activity of VWAP and Nacrein promoters. These results suggest that PmRunt may be directly involved in the regulation of VWAP and Nacrein gene expression. (2) MG4 and its homologous gene (C1qDC) regulated by PmRunt are involved in the formation of nacre in shells. MG4, as an effector gene downstream of PmRunt, belongs to the C1q domain containing protein (C1qDC) family. The function of PmRunt-regulated C1qDCs (Pma_318.183, Pma_576.501, Pma_10000069, Pma_10012374) in shell mineralization was studied. The results showed that although C1qDCs in both species exhibited evident dilatation, there was a high abundance of mantle expression in Pinctada martensii, suggesting that expanded C1qDCs participated in shell mineralization. The expression of Pma_318.183 (MG4), Pma_576.501 and PMA_10012374 in mantle envelope was significantly down-regulated by RNA interference technique (P 0.05). The formation of nacre on the inner surface of shells in three experimental groups was disordered by SEM. Therefore, C1qDCs may affect the formation of nacre in shells. (3) Ecdysterone may affect the formation of PmRunt and mineralized shells. In this study, the CDS sequence of ecdysone receptor and its binding protein RXR was validated. Elisa method was used to detect the changes of ecdysone content in serum after shell injury, and it was found that ecdysone was significantly involved in shell formation 2 hours after shell injury. Upregulation (P 0.05), the highest concentration at 4 h, and 36 h up-regulation was consistent with the appearance of shell repair membrane, suggesting that ecdysone may be related to shell repair. These results suggest that ecdysone may regulate the expression of PmRunt and other mineralization-related genes. 3. MicroRNA regulates the expression of PmRunt and NF-kappa B pathway genes. In this study, Pm-microRNAs were used to detect the regulation of Pm-microRNAs on PmRunt in vitro and in vivo overexpression of Pm-microRNAs-183. Luciferase activity was significantly down-regulated (P 0.05) after R-183 was co-transfected into 293T cells with pMireport vector containing 3 `UTR region of PmRunt; Pm-microRNA-183 expression was significantly up-regulated (P 0.05) and PmRunt expression was significantly down-regulated (P 0.05) after Pm miR-183 MICs analogue was injected into Mantle Tissues of Pinctada martensii. These results suggest that Pm-microRNA-183 may be involved in the regulation of nacre formation by interacting with PmRunt's 3'-UTR suppressor gene expression. Pm-microRNA-146a was obtained from Pinctada martensii by bioinformatics analysis, and the expression of Pm-microRNA-146a in different tissues was detected by stem-loop primer specific reverse transcription and qRT-PCR. The results showed that Pm-microRNA-146a was expressed in all tissues, higher in mantle tissues, and lower in blood. MicroRNAanda software predicted that Pm-microRNA146a and macrophage migration inhibitor (MIF) had a targeting effect. After injection of Pm-microRNA146a MICs analogue, the expression of Pm-microRNA146a in blood cells was increased. These results suggest that Pm-mi R-146 a may participate in the negative regulation of NF-kappa B pathway by regulating the expression of MIF.
【学位授予单位】：广东海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S917.4
，
本文编号：2185072
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