魁蚶(Scapharca broughtonii)血红蛋白Ⅰ基因克隆及表达特征

Cloning and Expression Analysis of Hemoglobin GeneⅠof Scapharca broughtonii

中文关键词: 魁蚶  血红蛋白  基因克隆  表达特征

英文关键词: Scapharca broughtonii  Hemoglobin  Gene clone  Expression characteristics

中文摘要:

      魁蚶(Scapharca broughtonii)的血淋巴中因含有血红蛋白而不同于其他多数双壳贝类。为明确魁蚶血红蛋白基因的结构特征、组织发生分布、免疫活性等特点，本研究以魁蚶转录组数据库中的部分序列为基础，通过cDNA末端快速扩增(Rapid amplication of cDNA ends, RACE)技术，克隆获得了一种魁蚶血红蛋白基因cDNA全长序列(命名为SbHbⅠ)，并研究了不同条件下的mRNA表达。序列和结构分析显示，SbHbⅠ基因cDNA全长为867 bp，其中包括长度为483 bp的开放阅读框(Open reading frame, ORF)，编码160个氨基酸，预测蛋白分子量为17.5 kDa，理论等电点为9.68；氨基酸序列具有多个血红素结合位点，序列与所选其他物种的相似度范围为57%~93%，与蚶科相似度较高。实时荧光定量PCR (qRT-PCR)结果显示，在魁蚶成体的斧足、鳃、外套膜、闭壳肌、血淋巴、肝胰腺及受精卵至发育23 d的幼虫体内，均能检测到SbHbⅠ基因；该基因在成体魁蚶血淋巴中表达量最高，与其他5个组织中的表达量差异显著(P<0.05)；受精卵至23 d幼虫期的表达，基本呈现为先下降后上升的趋势，且在12 d时急剧上升(P<0.05)；在鳗弧菌(Vibrio anguillarum)刺激和低氧胁迫后，血淋巴中SbHbⅠ基因的表达均呈先上升后下降的趋势，表达量最高值均出现在 16 h。本研究丰富了贝类血红蛋白相关研究资料。

英文摘要:

      Hemoglobin is the main component of haemolymph, which is widely distributed in vertebrates and some invertebrates. It has many biological functions, including transporting oxygen, storing energy, sustaining osmotic pressure, immunological functions, and so on. According to previous reports, the functions of hemoglobin from different organisms are varied, especially in invertebrate animals, because of less conservation in gene structure. However, the study of the immune function of hemoglobin in mollusks is limited. The ark shell, Scapharca broughtonii, is one of the important economic marine shellfishes in China, Korea, and Japan, and other places. In particular, previous studies showed that there were three types of hemocytes in S. broughtonii: red blood cells, white blood cells, and thrombus cells. To determine the structure, distribution, and immunological activity of the hemoglobin gene, the cDNA sequence of hemoglobin (named SbHbⅠ) was obtained using EST and RACE methods. Sequence analysis revealed that the cDNA was 867 bp in length, including an open reading frame (ORF) of 483 bp encoding a polypeptide of 160 amino acid residues. The predicted molecular weight was 17.5 kDa and isoelectric point (pI) was 9.68. The mRNA expression analysis of SbHbⅠin tested tissues by quantitative real-time PCR (qRT-PCR) revealed that the mRNA of SbHb I could all be detected in the foot, gill, mantle, adductor muscle, haemocytes, hepatopancreas, and the larvae aged from oosperm to 23 d. The expression level of SbHbⅠin haemocytes was more than 70 folds that in the adductor muscle. The expression of SbHbⅠin the larvae from the oosperm to 23 d was basically showed a declining tendency at the beginning and increased later, with a sharp increase at 12 d (P<0.05). After being stimulated in Vibrio anguillarum, SbHbⅠfirst showed an upgraded tendency and later descended, and the same tendency occurred during hypoxia stress, during which SbHbⅠreached the maximum (P<0.05) at 16 h at three dissolved oxygen levels (4.5 mg/L, 2.5 mg/L, and 0.5 mg/L). Basic data were provided from the study of hemoglobin in mollusks.