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| 昆虫双杂交实验中家蚕BmHSP83和BmMAD3蛋白引起的假性现象 |
| False phenomena of insect two-hybrid system caused by silkworm's proteins BmHSP83 and BmMAD3 |
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| DOI： |
| 中文关键词: 昆虫双杂交 假性 家蚕 HSP83 MAD3 |
| 英文关键词:Insect two-hybrid false phenomenon Bombyx mori HSP83 MAD3 |
| 基金项目:广东省科技计划项目（2016B020234005）；国家自然基金面上项目（31772184） |
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| 中文摘要: |
| 昆虫双杂交（Insect two-hybrid, I2H）技术是研究昆虫细胞蛋白质相互作用的一种方法。本研究利用Gateway克隆技术，构建了家蚕Bombyx mori蛋白BmHSP83和BmPDIA5，以及BmMAD3和BmMAN1的I2H载体；然后，将载体与报告质粒pUAS-Luc一起转染SF-9细胞，检测其发光值。结果表明，转染pIE2-AD-BmHSP83或pIE2-AD-BmMAD3载体与转染pUAS-Luc的对照组的细胞发光值无明显差异，报告基因未被激活；分别单独用pIE2-DBD-BmHSP83或pIE2-DBD-BmMAD3质粒转染细胞时，发光强度显著增加，报告基因被激活，说明BmHSP83和BmMAD3造成了假阳性结果；并且当细胞共转染pIE2-DBD-BmHSP83和pIE2-AD-BmPDIA5、pIE2-DBD-BmMAD3和pIE2-AD-BmMAN1时报告基因的激活反应显著提高，提示BmHSP83与BmPDIA5、BmMAD3与BmMAN1存在互作关系从而引起阳性反应，且与BmHSP83和BmMAD3的假阳性形成叠加效应；然而，当共转染pIE2-AD-BmHSP83和pIE2-DBD-BmPDIA5、pIE2-AD-BmMAD3和pIE2-DBD-BmMAN1时报告基因未被激活，显示pIE2-AD-BmHSP83/BmMAD3导致了假阴性。BmHSP83和BmMAD3引起I2H假性现象可能与该二种蛋白具有转录激活功能有关，当其与DNA结合结构域（DNA binding domain, DBD）融合表达时，该融合体即具备了DNA结合及转录激活两个功能，因而可以启动下游报告基因的表达；反之，当其与转录激活结构域（AD）融合表达时，则阻止了他们各自与BmPDIA5、BmMAN1蛋白的互作。本研究结果为那些利用I2H体系研究具有转录激活功能的蛋白质相互作用提供借鉴。 |
| 英文摘要: |
| Insect two-hybrid system (I2H) is a technology used to explore the interactions of proteins in insect cells. In current experiment, I2H vectors of silkworm (Bombyx mori) proteins, BmHSP83, BmPDIA5, BmMAD3 and BmMAN1 were constructed by means of Gateway cloning technology. Then, these I2H vectors with the reporter plasmid pUAS-Luc were transferred to SF-9 cells for fluorescence detection. The results indicated that the reporter gene was not activated when pIE2-AD-BmHSP83 or pIE2-AD-BmMAD3 was transfected in the cells, because the light intensity in treatments and control (pUAS-Luc transfected) were not significantly different. However, when respectively transfected with pIE2-DBD-BmHSP83 or pIE2-DBD-BmMAD3 plasmid, the light intensity of cells were increased significantly because the reporter gene was activated, which suggested that BmHSP83 and BmMAD3 cause I2H false-positives. Furthermore, the activation of reporter gene were significantly promoted after co-transfected by pIE2-DBD-BmHSP83 and pIE2-AD-BmPDIA5, or pIE2-AD-BmMAD3 and pIE2-DBD-BmMAN1, which suggested that BmHSP83 interacts with BmPDIA5, BmMAD3 interacts with BmMAN1, which leads to true positive reaction, and forms an additive effect with the false positive of BmHSP83 and BmMAD3. However, the reporter genes were not activated when the vectors, pIE2-AD-BmHSP83 and pIE2-DBD-BmPDIA5, or pIE2-AD-BmMAD3 and pIE2-DBD-BmMAN1 were co-transfected, which suggested that the pIE2-AD-BmHSP83 / BmMAD3 lead to false-negative. Probably, the I2H fake phenomena is related to the transcription function of BmHSP83 / BmMAD3. Therefore, the fusion proteins of the BmHSP83 / BmMAD3 and DNA binding domain (DBD) will have two functions of DNA binding and transcriptional activation, so that trigger the expression of downstream reporter genes. On the contrary, the fusion proteins of the BmHSP83 / BmMAD3 and transcriptional activation domain (AD)will inhibit the expression of downstream reporter genes. Our research provides new insights to I2H experiments of proteins with the function of transcriptional activation. |
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