厚壳贻贝两种新型防御素的分子鉴定

贻贝是全球范围内具有重要经济价值和生态价值的双壳贝类。贻贝抗菌肽具有极强的分子多样性,也是当前抗菌肽研究的重要对象。防御素是贻贝抗菌肽的重要成员, 从厚壳贻贝中鉴定到2种新型防御素, 但其分子特性和免疫机制尚不清楚。为此, 对厚壳贻贝体内新发现的2种防御素开展研究。序列分析结果表明,2种新型防御素均具有节肢动物防御素结构特征,因而被命名为arthropod like defensin (ALD)。利用荧光定量PCR研究了2种防御素在贻贝不同组织及不同发育阶段的表达量差异。进一步分析了2种防御素在3种不同微生物诱导下的表达量时间曲线。利用固相化学合成技术对2种防御素的成熟肽区进行合成并开展了功能验证。研究结果表明, 2种ALD 主要表达部位在外套膜和消化腺, 且ALD-1具有雄性特异表达特征。此外, ALD-1和ALD-2在贻贝幼虫阶段均未表达; 在不同微生物刺激下, 2种ALD表现出不同的免疫反应模式, 显示出2种防御素具有不同的免疫调节机制。化学合成的2种ALD均具有抑菌活性, 其对不同微生物的抑制率在20%~80%之间。上述研究为深入了解贻贝免疫防御的分子机制,以及贻贝抗菌肽的免疫功能和后续的分子资源开发奠定了基础。

Molecular Characterization of Two Novel Defensins from Mytilus coruscus

Mytilus is a bivalve species with important economic and ecosystem value over worldwide. Mytilus antimicrobial peptides, with strong molecular diversity, has become a research focus. Two novel antimicrobial peptides were identified from Mytilus, with structural features that similar to arthropod defensins. However, the functional features and the immune mechanism of these two mussel defensins are unknown. For this reason, the two novel defensins, Arthropod like defensn-1 and -2 (ALD-1 and ALD-2, respectively), were studied for the sequence features, expression profiles, and the dynamic expression pattern after different microbe induction. In addition, solid phase chemical synthesis technology was used for the synthesis of these two novel ALDs, and the function of synthesized peptides of ALD was verified. The results indicated that, two ALDs of M. coruscus have classical structure features similar to those of other arthropod defensins. These two ALDs are mainly presented in the tissues of mantle and digestive gland of mussel. The results also suggest that ALD-1 was expressed at higher levels in the gonads of males than in females (P<0.05). The expression of two ALDs is developmentally regulated,and both ALD-1 and ALD-2 were undetectable in larvae, but can be detected with high expression level at adult mussel with age of six months. The dynamic changes in the expression level of two ALDs after microbial induction were examined, and the results showed a marked increase in expression level observed in vivo for both ALDs. Interestingly, two ALDs showed different sensitivities to different microbes, indicating very complex responses during the mussel immune response. This observation strongly suggests the existence of different recognition mechanism or signal transduction pathway in mussels for the expression of ALD-1 and ALD-2. Moreover, both of two chemical synthesized ALDs showed significant antimicrobial activities against five tested microbes with an inhibition ratio of 20%-80%. These results provided basis for understanding the molecule mechanism of Mytilus immunology, and the function of novel Mytilus defensins, and thusly provided basis for the development of molecular resource for mussel antimicrobial peptides.