魁蚶（Scapharca broughtonii）C型凝集素基因家族的克隆与表达

发布时间：2018-07-05 14:40

本文选题：魁蚶 + C型凝集素　；参考：《上海海洋大学》2017年硕士论文

【摘要】：魁蚶(Scapharca broughtonii)俗称赤贝、血贝、大毛蚶,从属软体动物门(Mollusca),双壳纲(Bivalvia),翼形亚纲(Pterimorphia),蚶目(Arcoida),蚶科(Arcidae),是大型海洋底栖贝类的一种,经济价值很高,作为我国水产养殖领域的重要养殖对象,具有良好的市场前景。由于过度捕捞、环境污染以及不健康养殖导致病害发生,使魁蚶资源严重衰退。目前对于魁蚶的研究,主要集中在基础生物学以及养殖技术的研究,分子免疫方面对于魁蚶的报道不多,因此研究魁蚶免疫相关的分子机制,对于完善和丰富魁蚶的免疫学资料具有重要的意义。在454转录组测序的基础上,本文利用EST序列分析、cDNA末端快速扩增(RACE)技术从软体动物门的魁蚶中克隆获得四个凝集素基因cDNA全长序列(命名为Sb-Lec1、Sb-Lec2、Sb-Lec3、Sb-Lec4),并对这些基因进行了一定的功能分析,分析了四种基因在魁蚶中的组织分布以及在鳗弧菌(Vibrio anguillarum)胁迫下的mRNA表达量的响应机制并成功表达了Sb-Lec2蛋白,检测其对大肠杆菌、鳗弧菌、金黄色葡萄球菌的凝菌活性。具体结果如下:1.Sb-Lec1基因全长700bp,具有长度为504bp的开放阅读框,其编码167个氨基酸,其中信号肽序列的长度为23个氨基酸,129个氨基酸的糖识别结构域(CRD)以及参与二硫键形成的6个半胱氨酸。预测蛋白分子量为19.11kDa,理论等电点为4.74。多序列比对结果显示,Sb-Lec1基因CRD编码的氨基酸序列与其他物种的凝集素基因具有相似的结构,均含有形成二硫键的4个保守半胱氨酸。系统进化分析结果表明,魁蚶先与贝类等软体动物聚为一支,然后与脊椎动物聚在一起。采用荧光定量PCR技术,检测了Sb-Lec1在组织中的表达情况,结果显示该基因能够在肝胰腺、血淋巴、鳃、外套膜、闭壳肌、斧足组织中都表达,然而在肝脏中表达量最高。同时,分析了Sb-Lec1基因在鳗弧菌(Vibrio anguillarum)刺激下mRNA表达量变化情况。结果表明,相对于对照组,菌刺激组Sb-Lec1基因在所检测的每种组织中mRNA表达量都显著上调(P0.05),随着鳗弧菌暴露时间的延长,表达量一开始先升高,后来又出现降低的的趋势。2.Sb-Lec2基因全长756bp,具有489 bp的开放阅读框,能够编码162个氨基酸,预测该蛋白分子量的大小为18.73 kDa,理论等电点为4.49,编码一种分泌型蛋白,具有一个由135个氨基酸构成的CRD,该CRD含有的参与二硫键形成的6个半胱氨酸。系统分析结果表明,魁蚶Sb-Lec2在进化树上的位置是先与双壳贝类聚为一支,然后与鱼类,哺乳类聚为一类,与其分类地位是相似的。运用荧光定量PCR检测基因组织分布情况,结果显示,Sb-Lec2分布于所有被测组织,在肝胰腺表达量最高。鳗弧菌刺激后,相对于对照组,Sb-Lec2基因在所检测的每个组织中mRNA水平上的表达量都显著上调(P0.05),呈现先升高后降低的趋势。将Sb-Lec2基因进行原核重组蛋白表达,该重组蛋白表达形式是包涵体的形式,纯化、透析复性后对鳗弧菌、大肠杆菌、金黄色葡萄球菌都表现出抑菌活性,且对Ca2+无依赖性,对甘露糖无结合活性。3.Sb-Lec3基因和Sb-Lec4全长分别为692bp、643bp,开放阅读框长度分别为492bp和360bp,前者编码了163个氨基酸,后者编码了119个氨基酸,Sb-Lec3基因编码的蛋白质预测的蛋白分子量为18.96kDa,理论等电点为5.01,Sb-Lec4基因预测的蛋白分子量大小为12.37 kDa,理论等电点为5.80,两者编码的蛋白质都含有信号肽,为分泌型蛋白。多序列比对显示,这两种基因与其他物种的凝集素基因都具有一定的相似性,含有较为保守的半胱氨酸。系统进化树结果表明,两种基因编码的氨基酸序列都先与贝类聚为一类,在与其他物种聚为一支。这两种基因在正常个体组织中都有分布,且都在肝胰脏中分布最广,鳗弧菌刺激后,在不同组织中的变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.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S944.44

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