酚氧化酶级联途径在文蛤壳色—免疫关联中的作用研究

The clam Meretrix meretrix is an important commercial species in the coastal areas of China. The survival rates of three strains of clam M. meretrix with different color morphs obtained by selective breeding were investigated when a heavy mortality happened, and the results indicated that there was a significantly difference in the survival rate among these three strains. The phenomenon that more heavily melanised morph of M. meretrix had a higher survival rate implied that melanised morphs and immune response shared common molecular mechnism. Prophenoloxidase cascade (POC) has been considered to play an important role both in shell color formation and in the innate immune defense of M. meretrix, which made us believe that POC was the key molecular mechanism inducing the correlation between melanised morph and immunity of M. meretrix. In this study, firstly, we will verify how consistently, melanised shell color and immune defense of M. meretrix are correlated. Secondly, we will decipher the molecular basis of shell color variations of M. meretrix, which would help us understand the relationship between shell color and POC. Ultimately, we will analyze the effects of POC on the immune defense of clam and understand the relationship between them. Based on all results of our study, the relationship among shell color, immune and POC in M. meretrix will be sumarized, which would explain how and why clams with different melanised shell morphs displayed different degree of resistance. This study would complement our knowledge about the mechanism of innate immune defense of the bivalve, contribute to the furtherance of resistance selective breeding of M. meretrix, and provide informations for the quality evaluation and seed selection of other aquatic molllusc.

文蛤是我国重要的滩涂养殖贝类。我们在对选育获得的三个不同壳色文蛤品系进行养殖的过程中发现，爆发大规模死亡后，不同壳色文蛤品系的存活率存在显著差异,壳色越深，存活率越高。针对这一现象，本研究提出，文蛤壳色与免疫具有重叠的分子机制。鉴于酚氧化酶级联途径(prophenoloxidase cascade,POC)在文蛤的壳色形成与先天免疫中的双重作用，POC可能是产生不同壳色文蛤抗病力差异现象的关键。本研究在进一步明确不同壳色文蛤抗性差异程度的基础上，首先分析文蛤壳色多态的分子基础，解析文蛤壳色与POC的关系；然后分析POC在文蛤免疫中的作用，解析文蛤免疫与POC的关系；综合所有研究结果最终阐明文蛤壳色、免疫与POC三者的关系，解析文蛤壳色差异导致免疫差异的分子机制。预期研究结果将有助于了解贝类先天性免疫防御机制，为文蛤抗病新品种的选育提供科学依据，同时对其他水产贝类的种质评价与选种有借鉴意义。

文蛤是我国重要的滩涂养殖贝类。通过选择育种，我们获得了三个不同壳色的文蛤品系，在养殖过程中发现，爆发大规模死亡后，不同壳色文蛤品系的存活率存在显著差异,壳色越深，存活率越高。这一现象提示，文蛤壳色与免疫具有重叠的分子机制。鉴于酚氧化酶级联途径(prophenoloxidase cascade,POC)在文蛤的壳色形成与先天免疫中的双重作用，本项目开展了POC在文蛤壳色-免疫关联中的作用研究。首先，通过对三种壳色文蛤品系进行多次大规模弧菌攻毒试验、免疫相关酶活检测、弧菌抗性SNP检测等，进一步确认了红色壳文蛤品系表现出更强的弧菌抗性和免疫活力，具有一定的抗病力优势；同时，通过对不同壳色文蛤外套膜的光镜和电镜观察，明确了不同壳色文蛤外套膜的细微结构及特征差异，这些结果将为研究文蛤壳色形成的机制奠定基础。然后，克隆获得POC关键酶酚氧化酶、多巴脱羧酶、酪氨酸羟化酶的cDNA全长序列，并重组表达了相应蛋白，通过检测蛋白的抑菌活性以及在弧菌攻毒过程中的表达变化，明确了POC在文蛤弧菌免疫应答中的作用，从而阐明了文蛤免疫与POC的关系；通过定量PCR和原位杂交检测了POC关键酶在不同壳色文蛤组织中的表达差异，发现PO等表达在文蛤的外套膜和内脏团位置，其在不同壳色文蛤外套膜中的表达量存在显著差异，从而明确了文蛤壳色与POC的关系。最后，引入组学分析手段，分别从蛋白质组和转录组水平进一步证实了PO在不同壳色文蛤中的表达差异，也为相关研究的深入开展奠定数据基础。本项目的已有研究结果显示，在文蛤中壳色与免疫存在关联，并且这种关联能够用POC关键酶尤其是PO的表达差异来解释，本研究将有助于了解贝类先天性免疫防御机制，为文蛤抗病新品种的选育提供科学依据，同时对其他水产贝类的种质评价与选种有借鉴意义。