秀丽隐杆线虫经福尔马林处理后上调表达基因的分析

ANALYSIS OF UP-REGULATED GENES IN CAENORHABDITIS ELEGANS TREATED WITH FORMALIN

秀丽隐杆线虫(Caenorhabditis elegans)是一种重要的模式生物,目前已经被广泛应用到生物对各种驱虫剂抗性机制的研究中。福尔马林被普遍用于鱼类寄生虫病的防治中,但是由于长期的应用,许多寄生虫对它产生了一定的抗药性。研究以秀丽隐杆线虫为研究对象,分析了它在福尔马林刺激下基因表达的改变情况。结果显示,经福尔马林处理后差异表达的有676个克隆,通过斑点杂交技术进一步筛选,对其中差异显著的161个克隆进行了测序分析。测序结果经BLAST分析发现:(1)细胞凋亡相关基因的表达发生上调,这些基因编码包括线粒体呼吸链相关蛋白、含TPR序列的蛋白SGT-1、热休克蛋白、氧化应激相关蛋白、胞吞过程相关蛋白、DNA复制和修复相关蛋白以及其他一些重要的凋亡相关蛋白;(2)编码重要的转录调节因子和信号转导相关蛋白如转录激活因子FosB/c-Fos、G蛋白、细胞周期蛋白B、钙结合蛋白、核小体装配蛋白NAP-1等的基因的表达发生上调;(3)能量代谢和蛋白质、脂肪、氨基酸代谢途径中的有些基因的表达也发生上调;(4)除了以上已知功能的基因外,还有一些未命名蛋白质基因。这说明福尔马林对秀丽隐杆线虫的影响除了诱导细胞凋亡之外,还影响其他细胞代谢活动的改变。实验为进一步研究生物体对福尔马林的抗性机制奠定了一定的基础。

Caenorhabditis elegans, as a good model organism, has been used in the study of resistant mechanism against anthelmintics. On the other hand, formalin has been widely used in prevention and cure of fish parasites. However, many parasites generate resistance to formalin because of long term application. In the present study, techniques of suppression subtractive hybridization and dot blot hybridization were applied to analyze the gene expression alteration in C. elegans after formalin stimulation. C. elegans was serially cultivated in medium containing 800ppm formalin for one month. The worms growing in medium containing formalin was served as experimental group while those growing in normal medium as control group. Total RNA was extracted; mRNA was purified and reversely transcribed into cDNA. Forward suppression subtractive hybridization was used to deduct the cDNA contained in the control group out of the experimental group to enrich differential expression genes. Then, dot blot hybridization was used to further screen the up-regulation genes after formalin stimulation. The control cDNA and subtractive cDNA libraries were employed as probes, respectively, to hybridize with PCR product of the subtractive clones. It was found that 161 out of 676 gene clones showed evident expression discrepancy, and then they were sequenced after dot blot hybridization. BLAST analyses indicated the following genes up-regulated： （1） apoptosis related genes, encoding mitochondrial respiratory chain related proteins, protein SGT-1 containing TPR motif, heat shock proteins, oxidative stress related proteins, endocytosis related proteins, DNA duplication and reparation related proteins and other important apoptosis related proteins; （2） genes encoding important transcription regulation factor and signal transduction related proteins, for example, transcriptional activation factor FosB/c-Fos, G protein, cyclin B, Ca-binding protein, and nucleosome assembly protein NAP-1; （3） genes participating in energy, protein, fat and amino acid metabolisms; and （4） some genes encoding unnamed proteins. It indicates that, besides inducing apoptosis, formalin also impacts C. elegans’ cell metabolism.