三角帆蚌3个免疫相关基因的分子特征及表达分析

发布时间：2018-03-30 09:40

本文选题：三角帆蚌　切入点：免疫基因　出处：《上海海洋大学》2016年硕士论文

【摘要】：本研究以三角帆蚌(Hyriopsis cumingii)为研究对象,根据构建的三角帆蚌外套膜转录组文库中已标注的EST序列,应用cDNA末端快速扩增法(RACE)克隆了热休克蛋白90基因(HcHSP90)、活化的蛋白激酶C1基因(HcRACK1)和富含亮氨酸重复序列基因(HcLRR)的cDNA序列全长,对其进行了生物信息学分析,并分析了三个基因的组织表达及在不同应激条件下应激后不同时间点的表达。结果如下:1.HcHSP90基因的分子特征及表达分析HcHSP90基因cDNA全长为2659bp(GenBank登录号:KR633143),开放阅读框(ORF)为2187bp,另外其5'-和3'-非翻译区长度分别为101bp和371bp,其中3'非翻译区具有AATAAA mRNA加尾信号及Poly(A)寡聚腺苷酸。开放阅读框编码728个氨基酸,蛋白分子量为83.7Ku,等电点为5.13。序列中不存在信号肽和跨膜结构。氨基酸序列分析表明,HcHSP90含有HSP90家族5个保守的序列标签。同源比对分析显示,该基因编码的蛋白与褶纹冠蚌(Cristaria plicata)HSP90氨基酸序列的相似度最高,为83.93%。将所得的氨基酸序列与其他物种的HSP90的氨基酸序列进行多重序列比对,并构建系统树,结果显示,本实验克隆所得HcHSP90基因与已发表的几种软体动物HSP90聚为一支。在非胁迫状态下,HSP90基因在各个检测器官(闭壳肌、斧足、肝胰腺、血淋巴、外套膜、鳃、性腺)中均有表达,在肝胰腺中的表达量最高,性腺次之,闭壳肌中表达量最低。在大多数组织中,HSP90 mRNA表达水平与对照组(15℃)相比在不同温度处理(0,5,25,和35°C)均能显著被诱导。闭壳肌和性腺中,HSP90 mRNA表达水平在5℃达到高峰(8.89倍,P0.01;2.0倍,P0.01);肝胰腺中,HSP90mRNA在0℃下显著被抑制(0.29倍,P0.01);其余四种组织检测到HSP90mRNA表达水平均在35℃达到峰值。暴露于不同浓度的重金属镉(50,100,和200μg/L),HSP90 mRNA在血淋巴和鳃的表达水平上调程度不同,但没有明显的剂量依赖性反应。当嗜水气单胞杆菌侵染后,血淋巴中HSP90 mRNA表达上调,感染36h后达到高峰(12.14倍,P0.01),在鳃中,感染3h后表达显著上调(3.24倍,P0.01),然后保持恒定,直到感染36h后恢复至感染前表达水平。2.HcRACK1基因的分子特征及表达分析HcRACK1基因cDNA全长为1249 bp,其中开放阅读框957 bp,编码318个氨基酸。蛋白质结构域分析显示HcRACK1含有RACK1家族特有的7个WD40结构域。运用荧光定量PCR技术分析HcRACK1在非胁迫状态下及受到嗜水气单胞菌侵染和重金属镉暴露后相关组织表达量的变化。结果显示,HcRACK1在各个组织中均有表达,其中闭壳肌中表达量最高;嗜水气单胞菌侵染后,HcRACK1在血淋巴中的表达量逐渐上升,24 h时达到峰值,随后下降;暴露在100μg/L浓度的重金属镉中,HcRACK1在肝胰腺中的表达量逐渐上升,在第3d达到峰值;血淋巴在第2d达到峰值,随后下降;鳃中HcRACK1的表达量无明显变化。3.HcLRR基因的分子特征及表达分析HcLRR基因cDNA全长为3492 bp,其中开放阅读框2142 bp,编码713个氨基酸,包含一段由25个氨基酸组成的信号肽和688个氨基酸组成的成熟肽,氨基酸序列分析显示该序列存在跨膜结构,运用荧光定量PCR技术分析LRR在正常组织中及受到嗜水气单胞菌侵染后相关组织表达量的变化。结果显示,在非胁迫状态下,LRR基因在斧足中表达量最高,在其他组织中几乎不表达。嗜水气单胞菌刺激侵染后可以看到斧足和肝胰腺中LRR基因表达显著下调,而其他组织表达量在不同时间点均出现表达峰值,显示能显著被诱导。
[Abstract]:In this study, Hyriopsis cumingii (Hyriopsis cumingii) as the research object, according to the sequence of EST labeled Hyriopsis cumingii mantle transcriptome library construction in the application of rapid amplification of cDNA ends (RACE) clone the heat shock protein 90 gene (HcHSP90), protein kinase C1 gene activation (HcRACK1) and leucine rich repeat gene (HcLRR) full-length cDNA sequence, characterized by bioinformatics analysis, and analyzed the expression and expression of stress under different stress conditions at different time points after three gene organization. The results are as follows: analysis of full-length HcHSP90 gene cDNA 2659bp expression and molecular features of 1.HcHSP90 gene (GenBank accession No.: KR633143), the open reading frame (ORF) of 2187bp, the 5'- and 3'- untranslated region length were 101bp and 371bp, which has a AATAAA 3'untranslated region of mRNA signal and Poly (A) oligoadenylate open reading. Frame encoding 728 amino acids. The protein molecular weight is 83.7Ku and isoelectric point of signal peptide does not exist in the 5.13. sequence and transmembrane structure. The amino acid sequence analysis showed that HcHSP90 HSP90 family contains 5 conserved sequence tags. Homology analysis showed that the gene encoding the protein with c.plicata (Cristaria plicata HSP90) the amino acid sequence of the highest similarity to 83.93%. amino acid sequences obtained from other species of HSP90 multiple sequence alignment and phylogenetic tree, the results showed that the HcHSP90 gene and cloned in this experiment published several animal software HSP90 clustered into one clade. The stress in the presence of HSP90 gene in each detection of organ (axe foot, adductor muscle, hepatopancreas, hemolymph, mantle, gill, gonad) were expressed, expression in hepatopancreas is the highest, the second was the lowest in gonad and adductor muscle. In most groups The fabric, the expression level of HSP90 mRNA and the control group (15 C) were compared in different temperature treatment (0,5,25, and 35 C) were significantly induced. Adductor muscle and gonad, HSP90 mRNA expression level reached the peak at 5 degrees Celsius (8.89 times, 2 times, P0.01; P0.01); hepatopancreas. HSP90mRNA was inhibited significantly under 0 degrees (0.29 times P0.01); the other four tissues detected the expression level of HSP90mRNA reached the peak at 35 degrees Celsius. Exposure to cadmium in different concentrations (50100 and 200 g/L), HSP90 mRNA in the hemolymph of the gill and the expression level increased to different degrees, but no the reaction was dose dependent. When the Aeromonas hydrophila infection, expression of blood with Bazhong HSP90 mRNA, after 36h infection peaked (12.14 times P0.01), in the gills, significantly up-regulated after infection with 3H (3.24 times P0.01), and then kept constant, straight to the expression level of.2.HcRACK1 before infection gene recovery after 36h infection Analysis of full-length HcRACK1 gene cDNA was 1249 BP and the expression of molecular characteristics, including an ORF of 957 BP encoding 318 amino acids. The protein domain analysis showed that HcRACK1 contains 7 WD40 RACK1 motifs. Using fluorescence quantitative PCR analysis of HcRACK1 in non stress state and is addicted to the expression of related the organization of Aeromonas infection and cadmium exposure. The results showed that HcRACK1 expressed in all tissues, the expression of the adductor muscle was the highest; Aeromonas hydrophila infection, the expression of HcRACK1 in the hemolymph of the amount gradually increased and reached the peak of 24 h, then decreased; exposure to cadmium 100 g/L concentration in the expression of HcRACK1 in hepatopancreas weight increased gradually, and reached the peak at 3D and then decreased; hemolymph peak reached in 2D; the expression of HcRACK1 in gill had no significant change in.3.HcLRR gene Analysis of full-length HcLRR gene cDNA was 3492 BP and the expression of molecular characteristics, including an ORF of 2142 BP encoding 713 amino acids, containing a mature peptide composed of 25 amino acid signal peptide and 688 amino acids, amino acid sequence analysis showed that the sequence of transmembrane structure analysis of LRR in normal tissues and by the expression of related organization of Aeromonas hydrophila infection using fluorescence quantitative PCR. The results showed that the stress state in the presence of LRR gene in pelecypodium highest expression level, almost no expression in other tissues. Aeromonas hydrophila infection after stimulation can see the expression of pelecypodium and hepatopancreas the LRR gene was significantly down regulated, while other organizations expressed at different time points were shown to peak expression, was induced significantly.

【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S917.4

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