【摘要】：重組蕎麥胰蛋白酶抑制劑(recombinant buckwheat trypsin inhibitor,r BTI)是一種來源于蕎麥PotatoⅠ抑制劑家族的絲氨酸蛋白酶抑制劑,具有很好的生物活性及功能。先前的研究表明,r BTI在秀麗隱桿線蟲(Caenorhabditis elegans)中具有很好的延長壽命的性質(zhì),但其具體的作用機(jī)制還不太清楚。本文的研究證明,r BTI能夠調(diào)節(jié)轉(zhuǎn)錄因子DAF-16的轉(zhuǎn)錄活性,進(jìn)而影響線蟲的壽命,且該性質(zhì)與其胰蛋白酶抑制活性密切相關(guān)。通過定點(diǎn)突變技術(shù),分別對r BTI的45位、53位和44位氨基酸活性位點(diǎn)進(jìn)行突變,獲得了4種不同胰蛋白酶抑制活性的r BTI突變體,分別命名為r BTI-R45A,r BTI-R45F,r BTI-W53R和r BTI-P44T。經(jīng)典模式生物秀麗隱桿線蟲壽命檢測實(shí)驗(yàn)顯示,野生型r BTI可以明顯延長C.elegans的壽命,且在0~10μmol/L范圍內(nèi)具有濃度依賴性。和未處理對照組相比,10μmol/L野生型r BTI延長壽命幅度可達(dá)到14.5%,但是突變體r BTI-R45 A,r BTI-R45 F和r BTI-W53 R均不同程度失去了延長壽命的功能。利用熒光顯微觀察及qRT-PCR等方法進(jìn)一步研究發(fā)現(xiàn),野生型r BTI可增強(qiáng)壽命調(diào)控轉(zhuǎn)錄因子DAF-16的轉(zhuǎn)錄活性。與壽命檢測實(shí)驗(yàn)結(jié)果一致,4種r BTI突變體均不能使DAF-16轉(zhuǎn)錄活性增強(qiáng)。上述結(jié)果表明,在C.elegans中,r BTI可增強(qiáng)長壽因子DAF-16的轉(zhuǎn)錄活性,進(jìn)而延長蟲體壽命,且該功能的發(fā)揮依賴于其適當(dāng)?shù)囊鹊鞍酌敢种苹钚�。本文的結(jié)果為進(jìn)一步研究開發(fā)r BTI的功能提供了實(shí)驗(yàn)支持和理論基礎(chǔ)。
[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.
【作者單位】： 化學(xué)生物學(xué)與分子工程教育部重點(diǎn)實(shí)驗(yàn)室山西大學(xué)生物技術(shù)研究所;
【基金】：國家自然科學(xué)基金項目(No.31171659,No.31300653)~~
【分類號】：R151.41
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本文編號：2381195