基于多組學分析的馬氏珠母貝礦化相關基因研究
發(fā)布時間：2018-08-15 18:41
【摘要】：軟體動物貝殼是典型的生物礦化產(chǎn)物。馬氏珠母貝Pinctada fucata martensii是研究生物礦化的良好材料。本研究以課題組組裝獲得的馬氏珠母貝基因組精細圖為參考,通過比較基因組、蛋白質(zhì)組及轉(zhuǎn)錄組等多組學數(shù)據(jù)關聯(lián)分析,研究參與貝殼形成的基質(zhì)蛋白家族及其進化過程、發(fā)現(xiàn)與之相關的代謝通路和調(diào)控因子;利用RNA干擾、酵母雙雜交以及雙熒光素酶報告系統(tǒng)等多種研究方法對PmRunt上游信號分子、下游效應基因及其轉(zhuǎn)錄后調(diào)控因子的作用機制進行了研究。研究結果如下:1、組學分析發(fā)現(xiàn)礦化相關基因家族及調(diào)控因子(1)馬氏珠母貝基因組進化分析本研究對馬氏珠母貝、長牡蠣、帽貝、海蝸牛、章魚、小頭蟲、水蛭、斑馬魚和人九個物種基因組序列進行基因家族聚類并對擴張和收縮的基因家族進行分析。以軟體動物有殼類四個物種共同祖先與無殼的頭足類章魚的比較,發(fā)現(xiàn)貝殼形成的共同機制,即鈣離子信號通路及礦質(zhì)離子吸收、糖胺聚糖和多聚氨基糖生物合成及代謝為貝殼的形成提供了物種基礎。對馬氏珠母貝擴張收縮的基因家族進行分析,發(fā)現(xiàn)磺基轉(zhuǎn)移酶擴張與貝殼珍珠層中高豐度的酸性粘多糖的性狀相一致。對馬氏珠母貝和長牡蠣共同擴張的貝殼蛋白家族進行分析,發(fā)現(xiàn)類纖連蛋白(FNL)可能在二者貝殼形成中均發(fā)揮重要作用;酪氨酸酶家族(TYR)的擴張可能導致馬氏珠母貝貝殼酪氨酸酶蛋白獨立分支的出現(xiàn);含有VWA domain的蛋白家族(VWAP)可能對馬氏珠母貝珍珠層形成的貢獻力更大。對馬氏珠母貝特有基因家族進行分析,發(fā)現(xiàn)具有氨基酸偏好性和重復單元的特有貝殼蛋白家族RLCD(related low complexity domain)在馬氏珠母貝貝殼形成中發(fā)揮重要作用。(2)馬氏珠母貝發(fā)育及組織轉(zhuǎn)錄組分析本研究以馬氏珠母貝基因組精細圖為參考,對馬氏珠母貝發(fā)育及組織轉(zhuǎn)錄組樣品進行轉(zhuǎn)錄組測序和分析。(1)擔輪前期到擔輪期、眼點期到變態(tài)后時期貝殼蛋白表達明顯上調(diào),分別與貝殼原殼和次生殼的形成一致,提示這兩個階段是貝殼形成的重要時期。(2)除外套膜組織是貝殼形成的主要器官,血細胞和閉殼肌對礦化基因也具有一定貢獻。(3)對基因組進化分析獲得的馬氏珠母貝擴張的和特有的礦化相關基因家族表達模式進行分析,發(fā)現(xiàn)幾丁質(zhì)合成和代謝相關酶、磺基轉(zhuǎn)移酶、TYRs、VWAPs、FNLs以及RLCDs,均有部分基因在擔輪期或變態(tài)后時期表達量上調(diào),同時在外套膜組織或珍珠囊中高表達,提示基因家族的倍增和基因功能分化、以及礦化相關種系特有基因的出現(xiàn)可能在貝殼的形態(tài)進化及形成中發(fā)揮重要作用。(4)分析了已知礦化相關調(diào)控因子的表達模式,同時對兩個關鍵時期轉(zhuǎn)錄組差異表達基因進行功能富集,發(fā)現(xiàn)Wnt、BMPs、VEGF、MAPK、破骨細胞分化等信號通路以及Runt、SMAD、AP-1等轉(zhuǎn)錄因子可能在貝殼形成的調(diào)控中發(fā)揮重要作用;并且雌激素、維生素D3、甲狀腺素和蛻皮激素可能參與變態(tài)后次生殼的形成。2、轉(zhuǎn)錄因子PmRunt及相關通路調(diào)控珍珠層的形成(1)PmRunt對珍珠層形成的調(diào)控通過組學的分析篩選獲得可能參與貝殼形成的重要轉(zhuǎn)錄因子PmRunt,利用RACE技術對PmRunt進行了克隆,并對其配體PmCBF的CDS區(qū)進行了驗證。PmRunt基因全長2319bp,5`端非編碼區(qū)為82bp,3`端非編碼區(qū)為599bp,編碼的蛋白具有545個氨基酸。PmRunt含有典型的具有DNA結合能力的Runt結構域(RHD),-COOH端存在VWRPY modif。PmRunt與脊椎動物的RUNX1相似度最高,系統(tǒng)進化分析發(fā)現(xiàn)脊椎動物和無脊椎動物的Runt protein分為獨立的兩支,軟體動物的Runt protein來自同一祖先。酵母雙雜交結果證明PmRunt和PmCBP蛋白之間存在相互作用。為了研究PmRunt對貝殼形成的影響,本研究以注射dsRFP組為陰性對照、利用dsRNA介導的RNA干擾使得PmRunt的表達發(fā)生顯著性下調(diào)(P0.05),掃描電鏡檢測貝殼內(nèi)表面,發(fā)現(xiàn)珍珠層的形成發(fā)生紊亂,暗示PmRunt參與貝殼珍珠層的形成。轉(zhuǎn)錄組測序發(fā)現(xiàn)多個珍珠層基質(zhì)蛋白基因表達量在PmRunt干擾組發(fā)生顯著性下調(diào),其中包括carbonic anhydrase-like、TYR、VWAP、Mantle gene4(MG4)等。利用RO5-3335抑制劑抑制PmRunt與PmCBF的結合后,MG4(Pma_318.183和Pma_10000069)、VWAP(Pma_10019836)、TYR2(Pma_10029257)、Nacrein的表達量均發(fā)生顯著性下調(diào)(P0.05),暗示這些基因可能受到PmRunt的直接調(diào)控。因此,本研究利用雙熒光素酶報告系統(tǒng)檢測了PmRunt與VWAP(Pma_10019836)和Nacrein啟動子區(qū)域的相互作用,發(fā)現(xiàn)PmRunt可以增強VWAP和Nacrein啟動子的轉(zhuǎn)錄活性,提示PmRunt可能直接參與VWAP和Nacrein基因表達的調(diào)控。(2)PmRunt調(diào)控的MG4及其同源基因(C1qDC)參與貝殼珍珠層的形成MG4作為PmRunt下游的效應基因,屬于C1q domain containing protein(C1qDC)家族。本研究對馬氏珠母貝和長牡蠣發(fā)生擴張的C1qDCs的表達模式進行了分析,并對PmRunt調(diào)控的C1qDCs(Pma_318.183、Pma_576.501、Pma_10000069、Pma_10012374)在貝殼礦化中的功能進行研究。結果顯示,盡管兩個物種中C1qDC均出現(xiàn)明顯的擴張現(xiàn)象,但馬氏珠母貝中具有外套膜高豐度表達的集合,提示擴張的C1qDC參與貝殼礦化。利用RNA干擾技術顯著性下調(diào)Pma_318.183(MG4)、Pma_576.501和Pma_10012374在外套膜套膜區(qū)的表達(P0.05),SEM檢測發(fā)現(xiàn)3個實驗組的貝殼內(nèi)表面珍珠層的形成發(fā)生了紊亂,因此C1qDCs可能影響貝殼珍珠層的形成。(3)蛻皮激素對PmRunt及貝殼礦化相關基因表達的調(diào)控在發(fā)育轉(zhuǎn)錄組比較分析中,發(fā)現(xiàn)蛻皮激素可能參與貝殼的形成。本研究對蛻皮激素受體及其結合蛋白RXR的CDS序列進行了驗證。通過Elisa方法檢測貝殼損傷后血清中蛻皮激素含量的變化,發(fā)現(xiàn)蛻皮激素在缺殼后2h顯著性上調(diào)(P0.05),4h時的濃度最高,而36h的上調(diào)與貝殼損傷修復膜的出現(xiàn)一致,暗示蛻皮激素可能與貝殼的修復相關。利用40ng/L的蛻皮激素浸泡處理外套膜套膜區(qū)小片,轉(zhuǎn)錄因子PmRunt、AP-1、BMP2/7、VWAP、TYR2、KRMP和CHS的表達均發(fā)生顯著性變化(P0.05),以上結果提示蛻皮激素可能調(diào)控PmRunt的表達,同時也參與其他礦化相關基因的調(diào)控影響貝殼的形成。3、microRNA調(diào)控PmRunt及NF-κB通路基因表達本研究利用體外細胞實驗和Pm-miR-183活體過表達實驗檢測Pm-miR-183對PmRunt的調(diào)控作用,結果顯示Pm-miR-183與包含PmRunt的3`UTR區(qū)域的pmiRreport載體共轉(zhuǎn)染293T細胞后,熒光素酶活性發(fā)生顯著性下調(diào)(P0.05);注射PmmiR-183 mimics類似物后,馬氏珠母貝外套膜組織中的Pm-miR-183表達量發(fā)生顯著性上調(diào)(P0.05),PmRunt的表達量發(fā)生顯著性下調(diào)(P0.05),同時珍珠層的形成發(fā)生紊亂,以上結果表明Pm-miR-183可能通過與PmRunt的3`-UTR互作抑制基因的表達,參與珍珠層形成的調(diào)控。本研究利用生物信息學分析獲得馬氏珠母貝Pm-miR-146a,并利用莖環(huán)引物特異性反轉(zhuǎn)錄,qRT-PCR檢測Pm-miR-146a在不同組織的表達量,結果顯示Pm-miR-146a在各個組織中均有表達,在外套膜組織的表達量較高,在血液中的表達量較低。miRanda軟件預測發(fā)現(xiàn)Pm-miR-146a與巨噬細胞遷移抑制因子(MIF)存在靶向作用,注射Pm-miR-146a mimics類似物后,血細胞中Pm-miR-146a的表達量發(fā)生顯著性上調(diào),同時MIF和NF-κB的表達量發(fā)生顯著性下調(diào)(P0.05)。以上結果暗示Pm-mi R-146a可能通過調(diào)控MIF的表達參與NF-κB通路的負調(diào)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.
【學位授予單位】：廣東海洋大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S917.4
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本文編號：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.
【學位授予單位】：廣東海洋大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S917.4
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本文編號：2185072
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