本文選題：短裸甲藻毒素 + 巖沙�？舅���； 參考：《第二軍醫(yī)大學》2016年博士論文

【摘要】：海洋聚醚類毒素結(jié)構(gòu)新穎復雜,毒性強,穩(wěn)定性好,在全球分布廣泛。多來源于容易形成赤潮的有毒甲藻,通過食物鏈在貝類、魚類等海產(chǎn)品體內(nèi)富集,對于人類健康和海洋漁業(yè)造成了嚴重威脅�，F(xiàn)有的毒素檢測方法都存在各種各樣的不足,因此急需新型的快速檢測聚醚類毒素的方法。此外,由于天然海洋聚醚類毒素提取困難,甲藻基因組又很難解析,因此目前對于毒素的生物合成了解的比較匱乏。本課題以海洋聚醚類毒素中代表性的短裸甲藻毒素BTX、巖沙�？舅豍LTX和大田軟海綿酸OA為研究對象,從毒素的檢測和生物合成兩方面展開研究,主要研究內(nèi)容及結(jié)果如下:1.海洋聚醚類毒素BTX適配體的篩選與應用。采用體外SELEX技術(shù)經(jīng)過18輪的篩選富集,分別獲得了與海洋聚醚類毒素BTX-A和BTX-B特異性結(jié)合的高親和力適配體A5和B2。通過先混合靶標篩選后分開次級文庫篩選的方式,一次性獲得了兩種毒素同系物的適配體,建立了一種高效篩選同系物適配體的SELEX方法。在此基礎(chǔ)上A5和B2這兩個適配體序列進行了截短優(yōu)化和突變改造�；趍fold軟件對該適配體的二級結(jié)構(gòu)進行分析,截去A5引物區(qū)及5’端第一個莖環(huán),并進一步進行了非必須核苷酸的去除和突變,獲得僅含有32個核苷酸的核心序列A5-S3G(Kd=72n M)。且不與STX、GTX、BTX-B、PLTX、OA等毒素結(jié)合,具有較好的特異性。通過生物素-鏈霉親合素耦合在線制備了基于SSA芯片和BLI技術(shù)的適配體傳感器,建立了基于生物膜干涉適配體傳感器檢測BTX的方法。最佳檢測時間250 s,檢測范圍為100~2000 n M,最低檢測限4.5 n M(4 ng/ml),且無交叉反應,同時具有較低的變異系數(shù),可用于BTX-A的實驗室檢測。2.海洋聚醚類毒素PLTX適配體的篩選與優(yōu)化。采用體外SELEX技術(shù)經(jīng)過10輪的篩選富集,通過序列同源比對聚類分析,結(jié)合生物膜干涉技術(shù)對篩選得到的若干適配體序列進行親和力表征,獲得了6條與海洋聚醚類毒素PLTX特異性結(jié)合的高親和力適配體,適配體P-13親和力最高(Kd=0.9 n M)。在此基礎(chǔ)上,對P-5、P-13和P-18三條適配體序列進行了截短優(yōu)化�；趍fold二級結(jié)構(gòu)的分析,截去引物區(qū)及非必須核苷酸,獲得了2段與PLTX高親和力結(jié)合的核心序列,P-13S2(Kd=0.56 n M)和P-18S2(Kd=0.93n M),為PLTX生物傳感器的制備奠定了基礎(chǔ),提供了識別元件。將PLTX通過氨基偶聯(lián)到SPR CM5生物芯片表面,并用該芯片對優(yōu)化后的序列進行親和力表征,結(jié)合優(yōu)化后的序列及SPR技術(shù),該芯片可用于制備高靈敏的競爭法檢測PLTX的生物傳感器。3.基于轉(zhuǎn)錄組測序的OA生物合成相關(guān)基因的挖掘。選取了初始氮濃度:磷濃度分別為15:1和30:1的培養(yǎng)條件培養(yǎng)利瑪原甲藻P.lima,培養(yǎng)不同時間收集藻細胞進行轉(zhuǎn)錄組測序和de novo拼接,得到了98594個Unigene,其平均長度為1319 nt,N50為1856 nt。得到的轉(zhuǎn)錄本通過Nr數(shù)據(jù)庫、Uni Prot KB/Swiss-Prot數(shù)據(jù)庫、COG數(shù)據(jù)庫進行功能注釋及分類,通過KEGG數(shù)據(jù)庫進行相關(guān)代謝途徑注釋,其中73289個Unigene成功注釋到功能,注釋到333條代謝途徑。在此基礎(chǔ)上初步確定了代謝途徑中基因的上調(diào)及下調(diào)水平。最后利用RSEM計算每個轉(zhuǎn)錄本的RPKM值,并對相關(guān)代謝途徑中的轉(zhuǎn)錄本的表達差異進行了初步分析。在此基礎(chǔ)上初步解析了OA生物合成相關(guān)的重要基因,如編碼PKS、硫脂酶、黃素單加氧酶、環(huán)氧化酶、環(huán)氧化物水解酶、細胞色素P450等酶的基因在不同樣品中表達量的差異。綜上,本研究分別篩選出了能與BTX和PLTX高親和力、特異性結(jié)合的適配體,并對其進行優(yōu)化改造,成功制備了基于生物膜干涉技術(shù)的快速檢測BTX的適配體傳感器,獲得了能夠開發(fā)用于PLTX檢測的SPR芯片和適配體識別元件,對于聚醚類毒素檢測提供了新的方法和工具。此外,實現(xiàn)了對利瑪原甲藻P.lima的轉(zhuǎn)錄組解析,并比較了不同營養(yǎng)條件下培養(yǎng)不同時間利瑪原甲藻的基因的表達差異,在此基礎(chǔ)上初步解析了OA生物合成相關(guān)的重要基因的差異表達,對進一步闡明OA的生物合成途徑和表達調(diào)控以及對利瑪原甲藻的產(chǎn)毒進行遺傳改造奠定基礎(chǔ)。
[Abstract]:Marine polyether toxoid has a complex structure, strong toxicity, good stability and wide distribution in the world. It comes from toxic methania which is easy to form red tide, and is enriched in marine products such as shellfish and fish by food chain. There are serious threats to human health and marine fishery. There are all kinds of deficiencies in the existing methods of detection of toxins. Therefore, a new method for rapid detection of polyether toxoid is urgently needed. In addition, because the extraction of the natural marine polyether toxoid is difficult, the genome of the methatom is difficult to be analyzed. Therefore, the biosynthesis of the toxin is scarce. This topic is based on the representative short methanobacteria toxin BTX of the marine polyether toxoid, the sands sea anemone toxin PLTX and Soft cavernic acid OA is the research object, which is studied from two aspects of detection and biosynthesis of toxin. The main contents and results are as follows: 1. screening and application of marine polyether toxoid BTX aptamers. Using in vitro SELEX technology after 18 rounds of screening and enrichment, the specific binding of marine polyether toxoid BTX-A and BTX-B was obtained. High affinity ligands, A5 and B2., were selected by the screening of separate secondary libraries after the first mixed target screening. The aptamers of two toxin homologues were obtained at one time, and a SELEX method to efficiently screen the homologue aptamers was established. On this basis, the two aptamer sequences of A5 and B2 were truncated and mutated. Based on mfold The two stage structure of the aptamer was analyzed by the software, the A5 primer area and the first stem ring at the 5 'end were truncated, and the removal and mutation of non essential nucleotides were further carried out. The core sequence of only 32 nucleotides, A5-S3G (Kd=72n M), was obtained. It was not combined with STX, GTX, BTX-B, PLTX, OA and other toxins, which had better specificity. Through biotin - The aptamer sensor based on SSA chip and BLI technology is prepared online by streptavidin coupling. The method of detecting BTX based on the biofilm interferometric aptamer sensor is established. The optimum detection time is 250 s, the detection range is 100~2000 n M, the minimum detection limit is 4.5 n M (4 ng/ml), and there is no cross reaction, and has a low coefficient of variation, which can be used in B. The TX-A laboratory tests the screening and optimization of the.2. marine polyether toxoid PLTX aptamers. After 10 rounds of screening and enrichment by the SELEX technique in vitro, the affinity sequence of selected aptamers was obtained through the sequence homologous alignment cluster analysis and the biofilm interference technique, and 6 marine polyether toxoid PLTX was obtained. High affinity aptamers of heterosexual binding and aptamer P-13 have the highest affinity (Kd=0.9 n M). On this basis, the three aptamers of P-5, P-13 and P-18 are truncated and optimized. Based on the analysis of mfold two structure, the primer region and non essential nucleotides are truncated, and the core sequence of 2 segments with high affinity to PLTX is obtained, P-13S2 (Kd=0.56 n). And P-18S2 (Kd=0.93n M), which lays the foundation for the preparation of PLTX biosensors, and provides a recognition element. PLTX is coupled to the surface of the SPR CM5 on the surface of the biochip of the SPR CM5, and the affinity characterization of the optimized sequence is carried out with this chip. Combined with the optimized sequence and SPR technology, the chip can be used to prepare a highly sensitive competitive method for detecting PLTX. The biosensor.3. was based on the mining of OA biosynthesis related genes of the transcriptional sequence. The initial nitrogen concentration was selected: the phosphorus concentration was cultured in 15:1 and 30:1, respectively, to train the P.lima of OA, and to collect the algal cells for sequencing and de novo splicing at different time. 98594 Unigene were obtained, the average length was 1319 NT, N5. The transcriptional transcript obtained by 0 for 1856 nt. is annotated and classified through the Nr database, the Uni Prot KB/Swiss-Prot database, the COG database, and the related metabolic pathway annotations through the KEGG database, of which 73289 Unigene are successfully annotated to the function, annotated to 333 metabolic pathways. On this basis, the gene in the metabolic pathway is preliminarily identified. At last, the RPKM value of each transcript was calculated by RSEM and the difference in the expression of the transcriptional transcript in the related metabolic pathways was preliminarily analyzed. On the basis of this, the important genes related to OA biosynthesis, such as encoding PKS, sulfur lipase, flavin monooxygenase, cyclooxygenase, epoxide hydrolase, cytochrome P, were preliminarily analyzed. The difference in the expression of 450 isozyme genes in different samples. In this study, we screened out the aptamers with high affinity and specific binding to BTX and PLTX, and optimized them, and successfully prepared the aptamer sensor for rapid detection of BTX based on biofilm interference technology, and obtained the SPR that can be developed for PLTX detection. The microarray and aptamer identification elements provide new methods and tools for the detection of polyether toxoid. In addition, the transcriptional analysis of P.lima is realized and the differences in the expression of gene in different nutrition conditions at different time are compared. On the basis of this, the importance of OA biosynthesis is preliminarily analyzed. The differential expression of genes lays the foundation for further elucidate the biosynthetic pathway and expression regulation of OA and the genetic transformation of the toxin produced by dinoflagellate Lima.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：Q78

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本文編號：1892704