目的:通過3-溴丙酮酸（3-BrPA）處理秀麗隱桿線蟲（Caenorhabditis elegans）,觀察3-BrPA對線蟲的毒性和生存周期的影響。通過秀麗隱桿線蟲RNA干擾（RNAi）和突變株,分析3-BrPA對線蟲糖酵解途徑己糖激酶家族和藥物代謝的關鍵酶細胞色素P450 （cytochrome P450,CYP）家族的影響。創(chuàng)新點:首次報道了3-BrPA對秀麗隱桿線蟲有毒性作用,己糖激酶是3-BrPA對線蟲作用的重要靶點,而CYP-35A家族是線蟲代謝3-BrPA的主要酶類。方法:用不同濃度的3-BrPA處理秀麗隱桿線蟲,每24 h監(jiān)測一次存活率;用實時熒光定量聚合酶鏈反應（qPCR）檢測代謝相關基因的表達;通過RNAi沉默己糖激酶家族基因hxk-7、hxk-2和hxk-3;計算3-BrPA處理hxk家族RNAi株和細胞色素P450cym-35家族突變株后的致死中濃度(LC₅₀)。結論:3-BrPA對線蟲有明顯的毒性效應（圖1）;與對照組比較,3-BrPA處理組的線蟲平均壽命明顯縮短（圖2）;3-BrPA處理線蟲后hxk-1、hxk-2和hxk-3的信使RN... 

【文章來源】：Journal of Zhejiang University-Science B（Biomedicine & Biotechnology）. 2020,21(01)SCICSCD

【文章頁數(shù)】：10 頁

【文章目錄】：
1 Introduction
2 Materials and methods
    2.1 Cultivation of C.elegans
    2.2 Exposure of C.elegans to 3-BrPA
    2.3 Construction of HK gene molecular phyloge-netic tree
    2.4 Total RNA extraction and reverse transcrip-tion of C.elegans
    2.5 Determination of hxk-1,hxk-2,and hxk-3m RNA expression
    2.6 Silencing of the target genes hxk-1,hxk-2,and hxk-3
        2.6.1 Cloning the target genes
        2.6.2 Expression of the target genes
        2.6.3 Target gene silencing
        2.6.4 Design of CYP35 family primers and deter-mination of expression of cyp35 genes
    2.7 Action of 3-BrPA on RNAi or mutant nema-tode strains
    2.8 Data analysis
3 Results
    3.1 Effect of 3-BrPA on the life span of nematodes
    3.2 Evolutionary genetic comparison of the HK genes
    3.3 mRNA expression of the HK genes in C.ele-gans treated with 3-BrPA
    3.4 Effect of 3-BrPA on the HK genes in RNAi nematodes
    3.5 Effect of 3-BrPA on the expression of the cyp-35 gene in mutant strains
    3.6 LC50 of 3-BrPA on the cyp-35 gene mutant strains
4 Discussion
5 Conclusions
Contributors
Compliance with ethics guidelines



本文編號：3167635