重组荞麦胰蛋白酶抑制剂延长C.elegans寿命的作用机制
发布时间：2018-12-15 19:49
【摘要】：重组荞麦胰蛋白酶抑制剂(recombinant buckwheat trypsin inhibitor,r BTI)是一种来源于荞麦PotatoⅠ抑制剂家族的丝氨酸蛋白酶抑制剂,具有很好的生物活性及功能。先前的研究表明,r BTI在秀丽隐杆线虫(Caenorhabditis elegans)中具有很好的延长寿命的性质,但其具体的作用机制还不太清楚。本文的研究证明,r BTI能够调节转录因子DAF-16的转录活性,进而影响线虫的寿命,且该性质与其胰蛋白酶抑制活性密切相关。通过定点突变技术,分别对r BTI的45位、53位和44位氨基酸活性位点进行突变,获得了4种不同胰蛋白酶抑制活性的r BTI突变体,分别命名为r BTI-R45A,r BTI-R45F,r BTI-W53R和r BTI-P44T。经典模式生物秀丽隐杆线虫寿命检测实验显示,野生型r BTI可以明显延长C.elegans的寿命,且在0~10μmol/L范围内具有浓度依赖性。和未处理对照组相比,10μmol/L野生型r BTI延长寿命幅度可达到14.5%,但是突变体r BTI-R45 A,r BTI-R45 F和r BTI-W53 R均不同程度失去了延长寿命的功能。利用荧光显微观察及qRT-PCR等方法进一步研究发现,野生型r BTI可增强寿命调控转录因子DAF-16的转录活性。与寿命检测实验结果一致,4种r BTI突变体均不能使DAF-16转录活性增强。上述结果表明,在C.elegans中,r BTI可增强长寿因子DAF-16的转录活性,进而延长虫体寿命,且该功能的发挥依赖于其适当的胰蛋白酶抑制活性。本文的结果为进一步研究开发r BTI的功能提供了实验支持和理论基础。
[Abstract]:Recombinant buckwheat trypsin inhibitor (recombinant buckwheat trypsin inhibitor,r BTI) is a serine protease inhibitor derived from buckwheat Potato I inhibitor family. Previous studies have shown that, r BTI has the properties of prolonging life in (Caenorhabditis elegans), but its specific mechanism is not clear. It is proved that, r BTI can regulate the transcriptional activity of transcription factor DAF-16 and thus affect the longevity of nematodes, and this property is closely related to the inhibitory activity of trypsin. By site-directed mutagenesis, the amino acid active sites at 45, 53 and 44 positions of r BTI were mutated respectively. Four r BTI mutants with different trypsin inhibitory activity were obtained and named r BTI-R45A,r BTI-R45F,. R BTI-W53R and r BTI-P44T. The detection of the life of the classical model organism, Stereostrongylus elegans, showed that the wild-type r BTI could prolong the life span of C.elegans in a concentration dependent manner in the range of 0 ~ 10 渭 mol/L. Compared with the untreated control group, 10 渭 mol/L wild-type r BTI could prolong the life span by 14.5%, but the mutants r BTI-R45 ar BTI-R45 F and r BTI-W53 R lost the function of prolonging life in varying degrees. By fluorescence microscopy and qRT-PCR, it was found that wild-type r BTI could enhance the transcriptional activity of transcription factor DAF-16. In accordance with the results of life test, none of the four r BTI mutants could enhance the transcriptional activity of DAF-16. These results suggest that, r BTI can enhance the transcriptional activity of longevity factor DAF-16 in C.elegans, thus prolonging the longevity of the parasite, and the function depends on its proper trypsin inhibitory activity. The results of this paper provide experimental support and theoretical basis for further research and development of the function of r BTI.
【作者单位】： 化学生物学与分子工程教育部重点实验室山西大学生物技术研究所;
【基金】：国家自然科学基金项目(No.31171659,No.31300653)~~
【分类号】：R151.41
，
本文编号：2381195
[Abstract]:Recombinant buckwheat trypsin inhibitor (recombinant buckwheat trypsin inhibitor,r BTI) is a serine protease inhibitor derived from buckwheat Potato I inhibitor family. Previous studies have shown that, r BTI has the properties of prolonging life in (Caenorhabditis elegans), but its specific mechanism is not clear. It is proved that, r BTI can regulate the transcriptional activity of transcription factor DAF-16 and thus affect the longevity of nematodes, and this property is closely related to the inhibitory activity of trypsin. By site-directed mutagenesis, the amino acid active sites at 45, 53 and 44 positions of r BTI were mutated respectively. Four r BTI mutants with different trypsin inhibitory activity were obtained and named r BTI-R45A,r BTI-R45F,. R BTI-W53R and r BTI-P44T. The detection of the life of the classical model organism, Stereostrongylus elegans, showed that the wild-type r BTI could prolong the life span of C.elegans in a concentration dependent manner in the range of 0 ~ 10 渭 mol/L. Compared with the untreated control group, 10 渭 mol/L wild-type r BTI could prolong the life span by 14.5%, but the mutants r BTI-R45 ar BTI-R45 F and r BTI-W53 R lost the function of prolonging life in varying degrees. By fluorescence microscopy and qRT-PCR, it was found that wild-type r BTI could enhance the transcriptional activity of transcription factor DAF-16. In accordance with the results of life test, none of the four r BTI mutants could enhance the transcriptional activity of DAF-16. These results suggest that, r BTI can enhance the transcriptional activity of longevity factor DAF-16 in C.elegans, thus prolonging the longevity of the parasite, and the function depends on its proper trypsin inhibitory activity. The results of this paper provide experimental support and theoretical basis for further research and development of the function of r BTI.
【作者单位】： 化学生物学与分子工程教育部重点实验室山西大学生物技术研究所;
【基金】：国家自然科学基金项目(No.31171659,No.31300653)~~
【分类号】：R151.41
，
本文编号：2381195
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