视黄酸与长牡蛎黑色素生成的关系研究

Progress has been made in the breeding of black oyster, but the formation mechanism of oyster melanin is not clear. In previous work, no variations of tyrosinase genes were found related to shell color, but the transcription level of retinal dehydrogenase and tyrosinase gene in black oyster mantle were found significantly higher than that in white shell oysters. Based on the results of previous work, we proposed a hypothesis: more retinal dehydrogenase could catalysis retinal to produce more retinoic acid, retinoic acid combined with retinoic acid receptor and could form the complex of retinoic acid and retinoic acid receptor, the complex could combine the retinoic acid response element (RARE) to upregulate the expression of tyrosinase gene, and then more melanin was generated. In order to prove this hypothesis, we plan to first determine the retinal dehydrogenase gene can indeed affect the expression of oyster tyrosinase gene and the melanin deposition; then, determine the DNA sequence and location of RARE with ChIP-seq. Finally, in HEK293 cells, to verify if the combination of the retinoic acid-retinoic acid receptor complexes with the tyrosine enzyme promoter (including RARE) could promote Luciferase Reporter Gene expression after the promoter, so as to explain the molecular mechanism of the retinoic acid regulating the tyrosinase gene transcription and further affecting the melanin formation in Pacific oyster.

黑壳牡蛎的育种工作已取得进展，但牡蛎黑色素的生成机制尚不明确。在前期工作中，没有发现酪氨酸酶基因的编码区变异与壳色形成有关，但发现视黄醛脱氢酶和酪氨酸酶基因在黑壳牡蛎外套膜中的转录水平显著高于白壳牡蛎，基于前期工作和前人成果，我们提出假设：视黄醛脱氢酶表达上调后，催化产生更多的视黄酸，视黄酸将与视黄酸受体结合，形成"视黄酸-视黄酸受体"复合物，复合物会与酪氨酸酶基因调控区的视黄酸反应元件（RARE）结合，上调酪氨酸酶基因表达，然后生成更多的黑色素。为了证明此假设，我们计划首先确定视黄醛脱氢酶基因的确可以影响牡蛎酪氨酶基因表达和黑色素的生成；然后，确定RARE的序列及其位置，最后，在HEK293细胞中，验证视黄酸-视黄酸受体复合物与牡蛎酪氨酸酶启动子的RARE元件结合后，可否促进启动子之后荧光素酶基因的表达，从而探明视黄酸调控酪氨酸酶基因表达进而影响长牡蛎黑色素生成的分子机制。

牡蛎是世界上高产量的经济贝类，牡蛎贝壳颜色的研究对牡蛎的育种工作有着重要的指导意义。目前黑壳色牡蛎的育种工作已取得进展，但其中黑色素的生成机制尚不明确。在前期工作中，我们发现视黄醛脱氢酶和酪氨酸酶基因在黑壳牡蛎外套膜中的转录水平显著高于白壳牡蛎，暗示了视黄酸通路可能参与牡蛎的黑色素生成过程。.我们在长牡蛎中通过RNAi干扰视黄醛脱氢酶基因，荧光定量PCR检测后发现成功干扰后酪氨酸酶基因表达下调；在长牡蛎中注射外源视黄酸，通过转录组测序分析视黄酸对长牡蛎基因表达的影响，发现注射视黄酸后视黄酸受体基因RAR/RXR以及多个酪氨酸酶基因（TYR）表达激活。随后克隆视黄酸受体基因RAR和RXR并进行了组织分布、亚细胞定位及蛋白互作分析等功能验证。最后，验证了视黄酸受体的核受体功能，发现其可以结合视黄酸反应元件（RARE）组成的特定序列进而调控基因转录激活；筛选启动子区具有RARE的TYRs，检测RAR/RXR对TYR的调控作用，发现长牡蛎视黄酸受体RAR可以直接结合酪氨酸酶基因Tyr-2的启动子区并促进Tyr-2的转录激活。.另外，我们也对长牡蛎贝壳中黑色素的沉积机制进行了探究，发现牡蛎外套膜边缘存在三层褶皱，但只有外褶皱颜色与壳色一致，通过比较三层褶皱转录组，发现外套膜的三个褶皱存在功能分化。并且，在黑白条纹牡蛎中发现外套膜外褶皱边缘颜色与贝壳颜色存在对应关系。在外套膜外褶皱中筛选与另外两层褶皱差异表达的基因，为进一步探究牡蛎中黑色素形成机制提供了参考。