本文選題：魁蚶 + C型凝集素��； 參考：《上海海洋大學》2017年碩士論文

【摘要】：魁蚶(Scapharca broughtonii)俗稱赤貝、血貝、大毛蚶,從屬軟體動物門(Mollusca),雙殼綱(Bivalvia),翼形亞綱(Pterimorphia),蚶目(Arcoida),蚶科(Arcidae),是大型海洋底棲貝類的一種,經(jīng)濟價值很高,作為我國水產養(yǎng)殖領域的重要養(yǎng)殖對象,具有良好的市場前景。由于過度捕撈、環(huán)境污染以及不健康養(yǎng)殖導致病害發(fā)生,使魁蚶資源嚴重衰退。目前對于魁蚶的研究,主要集中在基礎生物學以及養(yǎng)殖技術的研究,分子免疫方面對于魁蚶的報道不多,因此研究魁蚶免疫相關的分子機制,對于完善和豐富魁蚶的免疫學資料具有重要的意義。在454轉錄組測序的基礎上,本文利用EST序列分析、cDNA末端快速擴增(RACE)技術從軟體動物門的魁蚶中克隆獲得四個凝集素基因cDNA全長序列(命名為Sb-Lec1、Sb-Lec2、Sb-Lec3、Sb-Lec4),并對這些基因進行了一定的功能分析,分析了四種基因在魁蚶中的組織分布以及在鰻弧菌(Vibrio anguillarum)脅迫下的mRNA表達量的響應機制并成功表達了Sb-Lec2蛋白,檢測其對大腸桿菌、鰻弧菌、金黃色葡萄球菌的凝菌活性。具體結果如下:1.Sb-Lec1基因全長700bp,具有長度為504bp的開放閱讀框,其編碼167個氨基酸,其中信號肽序列的長度為23個氨基酸,129個氨基酸的糖識別結構域(CRD)以及參與二硫鍵形成的6個半胱氨酸。預測蛋白分子量為19.11kDa,理論等電點為4.74。多序列比對結果顯示,Sb-Lec1基因CRD編碼的氨基酸序列與其他物種的凝集素基因具有相似的結構,均含有形成二硫鍵的4個保守半胱氨酸。系統(tǒng)進化分析結果表明,魁蚶先與貝類等軟體動物聚為一支,然后與脊椎動物聚在一起。采用熒光定量PCR技術,檢測了Sb-Lec1在組織中的表達情況,結果顯示該基因能夠在肝胰腺、血淋巴、鰓、外套膜、閉殼肌、斧足組織中都表達,然而在肝臟中表達量最高。同時,分析了Sb-Lec1基因在鰻弧菌(Vibrio anguillarum)刺激下mRNA表達量變化情況。結果表明,相對于對照組,菌刺激組Sb-Lec1基因在所檢測的每種組織中mRNA表達量都顯著上調(P0.05),隨著鰻弧菌暴露時間的延長,表達量一開始先升高,后來又出現(xiàn)降低的的趨勢。2.Sb-Lec2基因全長756bp,具有489 bp的開放閱讀框,能夠編碼162個氨基酸,預測該蛋白分子量的大小為18.73 kDa,理論等電點為4.49,編碼一種分泌型蛋白,具有一個由135個氨基酸構成的CRD,該CRD含有的參與二硫鍵形成的6個半胱氨酸。系統(tǒng)分析結果表明,魁蚶Sb-Lec2在進化樹上的位置是先與雙殼貝類聚為一支,然后與魚類,哺乳類聚為一類,與其分類地位是相似的。運用熒光定量PCR檢測基因組織分布情況,結果顯示,Sb-Lec2分布于所有被測組織,在肝胰腺表達量最高。鰻弧菌刺激后,相對于對照組,Sb-Lec2基因在所檢測的每個組織中mRNA水平上的表達量都顯著上調(P0.05),呈現(xiàn)先升高后降低的趨勢。將Sb-Lec2基因進行原核重組蛋白表達,該重組蛋白表達形式是包涵體的形式,純化、透析復性后對鰻弧菌、大腸桿菌、金黃色葡萄球菌都表現(xiàn)出抑菌活性,且對Ca2+無依賴性,對甘露糖無結合活性。3.Sb-Lec3基因和Sb-Lec4全長分別為692bp、643bp,開放閱讀框長度分別為492bp和360bp,前者編碼了163個氨基酸,后者編碼了119個氨基酸,Sb-Lec3基因編碼的蛋白質預測的蛋白分子量為18.96kDa,理論等電點為5.01,Sb-Lec4基因預測的蛋白分子量大小為12.37 kDa,理論等電點為5.80,兩者編碼的蛋白質都含有信號肽,為分泌型蛋白。多序列比對顯示,這兩種基因與其他物種的凝集素基因都具有一定的相似性,含有較為保守的半胱氨酸。系統(tǒng)進化樹結果表明,兩種基因編碼的氨基酸序列都先與貝類聚為一類,在與其他物種聚為一支。這兩種基因在正常個體組織中都有分布,且都在肝胰臟中分布最廣,鰻弧菌刺激后,在不同組織中的變化均是先升高后降低。
[Abstract]:Scapharca broughtonii, commonly known as red scallop, blood shellfish, big clam, Mollusca, Bivalvia, Pterimorphia, Arcoida, and clam (Arcidae), is a kind of large marine benthic shellfish, and has high economic value. It has a good market as a breeding object in the field of aquaculture in China and has a good market. Prospects. Due to overfishing, environmental pollution and diseases caused by unhealthy culture, the resources of the Quebec clam are seriously deteriorating. At present, the research on the clam mainly focuses on the research of basic biology and culture technology. There are few reports on the molecular immunity to the clam. On the basis of the sequencing of the 454 transcriptional group, we have cloned four lectin gene cDNA full-length sequences (named Sb-Lec1, Sb-Lec2, Sb-Lec3, Sb-Lec4) by EST sequence analysis and rapid amplification of cDNA terminal (RACE) technology from the clam of the mollusk gate. The tissue distribution of four genes in the subclam and the response mechanism of mRNA expression under the stress of Vibrio anguillae (Vibrio anguillarum) were analyzed and the Sb-Lec2 protein was expressed successfully. The specific results were as follows: the whole 1.Sb-Lec1 gene was 700bp, the specific results were as follows. An open reading frame with a length of 504bp encoded 167 amino acids in which the length of the signal peptide sequence is 23 amino acids, the 129 amino acid sugar recognition domain (CRD) and the 6 cysteine formed by the two sulfur bond. The predicted protein molecular weight is 19.11kDa, and the theoretical isoelectric point is shown by the 4.74. multiple sequence alignment, and the Sb-Lec1 gene CR D encoded amino acid sequences have similar structures with other species of lectin genes, all containing 4 conserved cysteines forming two sulfur bonds. Phylogenetic analysis showed that the clam was first clustered with molluscs and other mollusks and then together with vertebrates. The fluorescence quantitative PCR technique was used to detect Sb-Lec1 in the tissues. The results showed that the gene could be expressed in the hepatopancreas, hemolymph, gill, mantle, occult, and axe. However, the expression of the gene was highest in the liver. At the same time, the quantitative change of the expression of the Sb-Lec1 gene under the stimulation of Vibrio anguinae (Vibrio anguillarum) was analyzed. The results showed that, relative to the control group, the Sb-Lec1 based group was stimulated. The expression of mRNA was significantly up-regulated in each of the detected tissues (P0.05). With the prolongation of the exposure time of Vibrio Anguilla, the expression level began to rise first, and then the decreasing trend of the trend.2.Sb-Lec2 gene was fully 756bp, with a 489 BP open reading frame, which could encode 162 amino acids, and the molecular weight of the protein was predicted to be 18.73 kDa The theoretical isoelectric point is 4.49, encodes a secretory protein with a CRD of 135 amino acids, and the CRD contains 6 cysteines involved in the formation of the two sulfur bond. The result of systematic analysis shows that the position of the clam Sb-Lec2 in the tree of evolution first comes together with the bivalve shellfish, and then is grouped into a class of fish and breast-feeding, and is classified with it. The status was similar. The distribution of gene tissue was detected by fluorescence quantitative PCR. The results showed that Sb-Lec2 was distributed in all the tissues measured and expressed the highest in the hepatopancreas. After the stimulation of Vibrio Anguilla, the expression of the Sb-Lec2 gene on the level of mRNA in each of the tissues detected was up significantly (P0.05), showing a first rise and then descend. The Sb-Lec2 gene was expressed in the Prokaryotic Recombinant protein. The expression of the recombinant protein was the form of inclusion body. After purification and refolding, the recombinant protein showed bacteriostasis activity to Vibrio anguillae, Escherichia coli and Staphylococcus aureus, and was not dependent on Ca2+. The unbound mannose.3.Sb-Lec3 gene and the full length of Sb-Lec4 were 692bp, respectively. 643bp, the length of the open reading frame is 492bp and 360bp, the former encodes 163 amino acids, the latter encodes 119 amino acids. The protein molecular weight of the protein encoded by the Sb-Lec3 gene is 18.96kDa, the theoretical isoelectric point is 5.01, the protein molecular weight of the Sb-Lec4 gene is 12.37 kDa, the theoretical isoelectric point is 5.80, and the two are encoded. Protein contains a signal peptide, which is a secretory protein. Multiple sequence alignment shows that these two genes are similar to other species of lectin genes and contain more conserved cysteine. The phylogenetic tree shows that the amino acid sequences of the two genes are first together with shellfish and are together with other species. The two genes are distributed in the normal individual tissues and are most widely distributed in the hepatopancreas. After Vibrio Anguilla is stimulated, the changes in different tissues are all increased first and then decreased.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S944.44

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