Wang G-L, Xia X-L, Li X-L, Dong S-J, Li J-L
Key Laboratory of Freshwater Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China.
Key Laboratory of Freshwater Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai, China
Genet Mol Res. 2014 Jan 29;13(1):704-15. doi: 10.4238/2014.January.29.1.
Antimicrobial peptides (AMPs), of which big defensins are examples, are an important component of the natural defenses of most living organisms, and possess remarkable microbicidal activities. In the present study, using expressed-sequence tag sequences from a cDNA library and RACE, the full-length cDNA sequence of the big defensin gene from the triangle-shell pearl mussel, Hyriopsis cumingii, (HcBD), was cloned. The gene consists of a 5'-untranslated region (UTR) of 166 bp, a 3'-UTR region of 96 bp, and an open reading frame of 342 bp that encodes 113 amino acids, consisting of a 23 amino acid signal peptide and a mature peptide of 90 amino acids with a molecular mass of 12.5 kDa. Amino acid sequence analysis showed that the sequence contained a transmembrane domain and a hydrophobic region. The full-length amino acid sequence showed the highest similarity to an amphioxus (Branchiostoma floridae) sequence (64%), and lower similarities to other known defensins (α-, β-, and θ-defensins, and insect defensins). Expression of HcBD was relatively high in the mantle and blood, lower in other tested tissues, and absent in gill and foot tissues. Real-time quantitative PCR was used to investigate HcBD expression in various tissues at different time points after injection of Aeromonas hydrophila. At 4 h post-inoculation, HcBD expression in the mantle, liver, intestine, gill, and foot was greater than in the control, with the greatest expression at 72 h, while at 24 h, expression in the liver, intestine, gill, and foot were at their lowest levels. These results suggest that HcBD might play an important role in the host immune response. This study enriches the basic research on the big defensin family of antimicrobial peptides and lays foundations for further research on antimicrobial peptide expression and relevance to disease defense.
抗菌肽（AMPs），其中大防御素就是例子，是大多数生物体天然防御的重要组成部分，并具有显著的杀菌活性。在本研究中，利用来自cDNA文库的表达序列标签序列和RACE技术，克隆了三角帆蚌（Hyriopsis cumingii）大防御素基因（HcBD）的全长cDNA序列。该基因由一个166bp的5'-非翻译区（UTR）、一个96bp的3'-UTR区和一个342bp的开放阅读框组成，该开放阅读框编码113个氨基酸，包括一个23个氨基酸的信号肽和一个90个氨基酸的成熟肽，分子量为12.5kDa。氨基酸序列分析表明，该序列包含一个跨膜结构域和一个疏水区域。全长氨基酸序列与文昌鱼（Branchiostoma floridae）序列的相似性最高（64%），与其他已知防御素（α-、β-和θ-防御素以及昆虫防御素）的相似性较低。HcBD在蚌体外套膜和血液中的表达相对较高，在其他检测组织中较低，在鳃和足部组织中未检测到。利用实时定量PCR研究了注射嗜水气单胞菌后不同时间点各组织中HcBD的表达情况。接种后4小时，外套膜、肝脏、肠道、鳃和足部中HcBD的表达高于对照组，在72小时时表达最高，而在24小时时，肝脏、肠道、鳃和足部中的表达处于最低水平。这些结果表明，HcBD可能在宿主免疫反应中发挥重要作用。本研究丰富了抗菌肽大防御素家族的基础研究，为进一步研究抗菌肽表达及其与疾病防御的相关性奠定了基础。
