中文摘要：

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。

英文摘要：

Recombinant buckwheat trypsin inhibitor（ r BTI） has a variety of important bioactivities and functions. r BTI is a member of serine protease inhibitor of the potato inhibitor I family. Previous study showed that r BTI could extend lifespan in Caenorhabditis elegans（ C. elegans）,but its mechanisms remained unclear. In this paper, we demonstrated that r BTI extended lifespan by enhancing the transcriptional activity of DAF-16,and this effect was relevant with its trypsin inhibitory activity. The amino acid residues 45,53 and 44 of r BTI were mutated. r BTI-R45 A,r BTI-R45 F,r BTI-W53 R,and r BTI-P44 T mutants were generated by means of site directed mutagenesis. The lifespan assay ofC. elegans showed that r BTI significantly extended the lifespan in a dose-dependent way within the range of experimental doses（ 0 - 10 μmol/L）. Compared with worms treated without r BTI,the mean lifespan was extended by 14. 5 % in worms treated with 10 μmol/L r BTI. However,the mutants r BTI-R45 A,r BTI-R45 F and r BTI-W53 R lost the property of prolonged lifespan at different levels. Furthermore,fluorescence microscopy and qRT-PCR showed that r BTI increased the transcriptional activity of DAF-16,which is the main transcriptional factor regulating lifespan in C. elegans. Consistent with the study of lifespan,the mutants of r BTI also did not increase the transcriptional activity of DAF-16. Overall,these results suggested that r BTI increased longevity factor DAF-16 transcriptional activity, and extended the lifespan which both were closely related to its trypsin inhibitory activity. These finding provided experimental support and theoretical basis for the further research and development of r BTI function.