家蚕丝腺不同部位pH差异对i-motif结构的形成及其所调控基因转录的影响

DNA i-motif是一种在酸性条件下由半质子化的C·C+碱基对堆积而成的高级结构。通过调节细胞内pH至酸性可以促进i-motif结构的形成。我们前期发现，POUM2基因启动子区域i-motif结构的形成促进POUM2的表达。然而，i-motif结构是否能够响应环境pH变化进而影响其所调控基因的表达仍需进一步研究。而家蚕前、中及后部丝腺在生理条件下分别呈酸、中及碱性，POUM2基因在前部丝腺中的表达水平显著高于中、后部丝腺。因此，本项目以家蚕丝腺为材料，通过分析丝腺中i-motif的分布特征、调节丝腺离体培养时的pH值及敲除POUM2启动子上i-motif结构、用特异性结合i-motif结构的化合物处理等手段，探索i-motif结构是否能够响应细胞pH的变化进而调控POUM2等基因在丝腺不同部位的差异表达。本项目为进一步深入认识和丰富DNA高级结构调控基因差异表达的分子机制提供依据。

DNA is one kind of secondary sturctures formed via a stack of intercalating neutral C- hemiprotonated C base pairs (C·C+) under acidic condition. It is beneficial to the formation of the i-motif structure by changing the pH in cells to acidity. We previously identified an i-motif structure in the promoter region of BmPOUM2 gene and the formation of i-motif facilitated BmPOUM2 expression in the silkworm, Bombyx mori. However, the mechanism that i-motif structures could response to the changes of cellular pH and then affect the expression of genes still need further studies. The silk gland is consist of anterior, middle and posterior silk gland, which are acidic, neutral and alkaline respectively. The expression level of BmPOUM2 in anterior silk gland was significantly higher than that in middle and posterior silk glands. This project will take the silkworm silk gland as the research material to explore whether the i-motif structure regulates the differential expression of POUM2 in different parts of the silk gland by responding to different pH in silk gland cells. This project will take following measures: 1) analyze the structural distribution characteristics of i-motif in the silk gland; 2) change the pH value of the culture medium of the silk gland in vitro and 3) knockout the i-motif structure in the POUM2 promoter as well as TMPyP4 treatment, to clarify that the differential expression of genes including BmPOUM2 are due to the formation or deformation of i-motif structure in different pH conditions. This project provides a basis for further understanding and enriching the molecular mechanism of differential expression of genes regulated by DNA higher structures.