基质蛋白在珍珠和贝壳的形成过程中具有重要的作用，研究TGFβ信号通路对其表达的调控作用，将有助于从细胞和分子水平揭示珍珠形成的机理及信号转导途径。本课题拟从合浦珠母贝体内克隆TGFβ信号通路中几种关键蛋白的同源编码基因，包括受体蛋白Smad5、辅助因子Smad4、拮抗因子Smad6以及软骨形成相关蛋白Sox9、Sox5，进而研究TGFβ信号对珍珠和贝基质蛋白表达的调控作用。刺激或抑制Smad3和Smad5，检测不同生长状态下各转录因子和基质蛋白mRNA表达的变化情况，从而鉴定可能受到TGFβ信号调控的基质蛋白。重点以基质蛋白KRMP为对象，利用CHIP和EMSA等实验研究Sox9蛋白与其启动子的结合位点；利用荧光素酶报告基因载体（pGL3）研究Smad3、Smad5及Sox9对KRMP启动子转录活性的调节作用，以期探明TGFβ信号通路调控KRMP蛋白表达的机制和信号传导途径。

Molluscan matrix proteins are considered to play important roles in pearl and shell formation. Investigating the effect of the TGFβ signaling pathway on matrix proteins expression would help us to elucidate the mechanism and signaling pathway involved in pearl formation on cellular and molecular level. Here, we intend to clone the homologous genes of several important TGFβ signaling pathway proteins, including receptor Smad5, co-receptor Smad4, inhibitroy Smad6 as well as cartilage formation related protein Sox9 and Sox5, from the pearl oyster, Pinctada fucata，which may contribute to investigate the effect of the TGFβ signaling pathway on regulating the expression levels of those pearl and shell formation related matrix proteins. To identify the matrix proteins probably regulated by TGFβ signals, we intend to analyze the mRNA expression levels of the potential transcription factors and matrix proteins after the receptor smad proteins (Smad3 or Smad5) enhancement as well as inhibition. In this study, we investigate the binding affinity of Sox9 and the promoter of KRMP by both chromatin immunoprecipitation assay (CHIP) and Electrophoretic mobility shift assays (EMSAs). Meanwhile, we intend to study the effect of transcription factors such as Smad3, Smad5 and Sox9 on regulating the transcription activity of the promoter of KRMP by luciferase reporter assays. These results would help to elucidate the particular mechanism and TGFβ signaling pathway on regulating the expression level of matrix protein KRMP.