褶纹冠蚌(Cristaria plicata)样本壳长(9.50±1.05) cm、壳高(6.45±0.81) cm,暂养1周。40只随机分成试验组和对照组,每组20只。用Trizol 法提取经诱导刺激后0、6、12、24、36、48 h的血液、外套膜、鳃、肝胰腺组织RNA,构建褶纹冠蚌cDNA文库并从中获得防御素基因cDNA全长序列。结果表明,褶纹冠蚌防御素序列全长为514 bp,其中5’ UTR 有57 bp；3’ UTR 有242 bp,含有一个poly(A)尾；开放阅读框长度为192 bp,编码63个氨基酸组成的蛋白质,该蛋白 N 端为1个由23个氨基酸构成的信号肽,成熟肽由40个氨基酸组成,理论等电点为8.72,分子量为7.13 kD。三级结构预测显示其由一个α螺旋和两个β折叠片组成,形成典型的 CSαβ结构。通过注射109 cell/mL 的嗜水气单胞菌(Aeromonas hydrophila)刺激褶纹冠蚌,半定量分析PCR对褶纹冠蚌血液、外套膜、鳃和肝胰腺组织的防御素基因表达,结果表明,诱导前防御素基因在外套膜组织中表达量最高,约为血液和鳃的1.25倍,肝胰腺次之。在诱导0、6、12、24、36、48 h后,对照组的防御素基因表达量在血液和鳃中无明显变化,而在外套膜和肝胰腺中则呈现先略有下降而后上升；试验组防御素基因在血液中12 h 时表达量最高,随后略有下降,而在外套膜、鳃和肝胰腺组织中表达量均在0h 时出现峰值,而后呈现下降趋势；外套膜和鳃的表达量分别在24 h 和36 h 略有上升。本研究通过分析褶纹冠蚌防御素基因全序列,并利用嗜水气单胞菌对褶纹冠蚌进行注射感染,探讨防御素基因的表达规律,以期为防御素对贝类非特异性免疫的影响提供基础依据。%The specimen of Cristaria plicata with shell length(9.50±1.05) cm and shell height(6.45±0.81) cm were temporally cultured for one week. We designed test trial and control trial with 20 individuals for each. We obtained the full-length cDNA sequence of defensin from the cDNA library of Cristaria plicata. The full-length cDNA was 514 bp, consisting of a 5'-terminal untranslated region(UTR)of 57 bp, a 3'-terminal UTR of 242 bp with a poly(A)tail, and an open reading frame of 192 bp which encoded a polypeptide of 63 amino acids residues, in-cluding 23 signal peptides and 40 mature peptides. The predicted molecular weight was 7.13 kD, the theoretical isoelectric point was 8.72. The predicted structure revealed that defensin gene contained a conserved cys-teine-stabilized α-helix and two β-sheet(CSαβ)structural motif. We measured the expression defensin in the hemocytes, mantle, gill, and hepatopancreas of C. plicata by semi-quantitative RT-PCR following stimulation by Aeromonas hydrophila(109 cell/mL). Before stimulation, the expression of defensin was highest in the mantle and was about 1.25 times that in hemocytes and gill. After injection(at 0, 6, 12, 24, 36, 48 h), the relative expression first decreased in the mantle and hepatopancreas then increased significantly in the control group. There was no change in defensin expression in the hemocytes and gill. In the experimental group, expression peaked after 12 h in the hemocytes then decreased. Conversely, defensin expression was highest in the other tissues at 0 h, and then decreased gradually. Expression increased slightly in the mantle and gill at 24 h and 36 h.
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