Shell color as an important economic trait is also the crucial target trait for breeding and production. MicroRNA (miRNA) is an endogenous small non-coding RNA that can post-transcriptionally regulate the expression of target genes, it plays important roles in many life activities and physiological processes, such as shell color, stress response, and disease traits. In this study, we investigated the function of lgi-miR-2d in shell melanin formation and the expression patterns of lgi-miR-2d and target gene Rpmitf in Manila clam Ruditapes philippinarum. We further explored and verified the relationship between Rpmitf and lgi-miR-2d and identified the expression level of shell color-related gene changes by RNAi and injecting the antagomir of lgi-miR-2d, respectively. Our results indicated that lgi-miR-2d antagomir affected the expression of its target gene Rpmitf. In addition, the dual-luciferase reporter assay was conducted to confirm the direct interaction between lgi-miR-2d and Rpmitf. The results showed that the expression levels of melanin-related genes such as Rpmitf and tyr were significantly decreased in the positive treatment group compared with the blank control group after the Rpmitf dsRNA injection, indicating Rpmitf plays a crucial role in the melanin synthesis pathway. Taken together, we speculated that lgi-miR-2d might be negatively modulating Rpmitf, which might regulate other shell color-related genes, thereby affecting melanin synthesis in R. philippinarum.

壳色作为重要的经济性状，也是育种和生产的重要目标性状。 MicroRNA（miRNA）是一种内源性小非编码RNA，可以转录后调节靶基因的表达，在贝壳颜色、应激反应、疾病性状等许多生命活动和生理过程中发挥着重要作用。在本研究中，我们研究了lgi-miR-2d在菲律宾蛤仔贝壳黑色素形成中的功能以及lgi-miR-2d和靶基因Rpmitf在马尼拉蛤中的表达模式。我们进一步探索和验证了Rpmitf和lgi-miR-2d之间的关系，并分别通过RNAi和注射lgi-miR-2d的antagomir鉴定了壳颜色相关基因变化的表达水平。我们的结果表明 lgi-miR-2d antagomir 影响其靶基因Rpmitf的表达。此外，进行双荧光素酶报告基因测定以确认 lgi-miR-2d 和Rpmitf 之间的直接相互作用。结果显示，注射Rpmitf dsRNA后，阳性治疗组与空白对照组相比， Rpmitf、tyr等黑色素相关基因的表达量显着降低，表明Rpmitf在黑色素合成途径中发挥着至关重要的作用。综上所述，我们推测 lgi-miR-2d 可能负向调节Rpmitf，从而调节其他贝壳颜色相关基因，从而影响菲律宾R. philippinarum黑色素的合成。