大肠杆菌内源性非编码RNA+OxyS对秀丽线虫che-2基因表达与行为的调控

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1、目录中文摘要························································1ABSTRACT························································4前言·························································7材料与方法·····················································8结果···················

2、······································21讨论·························································29结论·························································33参考文献·······················································34综述···········································

3、·················37参考文献·······················································43致谢·························································45华中科技大学硕士学位论文大肠杆菌内源性非编码RNAOxyS对秀丽线虫che-2基因表达与行为的调控硕士研究生：吴金晶指导老师：王学仁副教授华中科技大学同济医学院附属同济医院麻醉学教研室中文摘要目的：研究大肠杆菌内源性非编码RNAOxyS对取食大肠杆菌的秀

4、丽线虫化学感受基因che-2的表达和秀丽线虫行为的调控，以及这种调控作用的分子机理。方法：通过秀丽线虫行为学实验分析大肠杆菌内源性非编码RNAOxyS对秀丽线虫行为的影响；逆转录PCR(PolymeraseChainReaction，聚合酶链反应)和实时定量PCR分别对秀丽线虫的化学感受基因che-2的mRNA表达进行半定量和定量的对比；通过蛋白质印迹（WesternBlot）对秀丽线虫的CHE-2::GFP蛋白进行定量对比；使用共聚焦显微镜对秀丽线虫体内具有荧光标记的che-2基因时空表达进行成像研究。结果：1.食物竞争实验：O

5、xyR和OxyS细菌上的野生型秀丽线虫N2的线虫数量随着时间逐渐减少，而ΔOxyS细菌上的线虫数量无明显变化。OxyS细菌上减少的线虫数量多于OxyR细菌。食物寻找能力实验：90min内95%的野生型N2能够找到食物OP50细菌，持续喂食OxyR或OxyS细菌一周后的野生型N2线虫能找到食物的线虫数量明显减少，分别为80%和75%，而且喂食OxyS细菌者比喂食OxyR细菌者更少。而只有大约10%的che-2突变型线虫能够找到食物OP50细菌。2.氯化钠趋化实验：90min内95%的野生型N2能够到达氯化钠液滴，持续喂食OxyR或O

6、xyS细菌一周后的野生型N2能够找到氯化钠液滴的线虫数量明1华中科技大学硕士学位论文显减少，分别为40%和25%，而且喂食OxyS细菌者比喂食OxyR者更少。只有大约10%的che-2突变型线虫能够找到氯化钠。醋酸钠化学躲避实验：90min内5%的野生型N2穿过醋酸铜线到培养盘的另一端，持续喂食OxyR或OxyS细菌一周后的野生型N2穿过醋酸铜线的数量明显增加，分别为20%和35%，而且喂食OxyS细菌者比喂食OxyR者更多。大约45%的che-2突变型线虫穿过醋酸铜线。3.食物竞争实验：野生型N2在OxyS细菌上的线虫数量随着时

7、间逐渐减少，但在OxyS-cb细菌上无明显变化。C.briggsae线虫在OxyS-cb细菌上的线虫数量随着时间逐渐减少，但在OxyS细菌上无明显变化。4.食物竞争实验：野生型N2在OxyR细菌上的线虫数量随着时间逐渐减少，alg-2，sid-1，sid-2突变型线虫在OxyR细菌上的线虫数量也减少，dcr-1，rde-1，rde-2突变型线虫减少的程度相对较小，而alg-1，rde-4突变型线虫减少的程度最小。5.食物竞争实验：野生型N2在OxyS细菌上的线虫数量会随着时间明显减少，而在che-2RNAi1细菌，che-2RNA

8、i2细菌上的线虫数量减少的程度相对较小。食物寻找能力实验：90min内95%的野生型N2能够找到食物OP50细菌，喂食OxyS细菌、che-2RNAi1细菌或che-2RNAi2细菌一周后的野生型N2找到食物的线虫数量明显减少，分别为60%，75%