秀丽隐杆线虫感知吸引性信号分子吲哚和2-乙基己醇的信号途径

发布时间：2018-05-19 18:19

本文选题：趋化性 + 吲哚　；参考：《云南大学》2015年硕士论文

【摘要】：模式生物秀丽隐杆线虫生活在复杂的土壤环境中,它以土壤中的细菌作为食,同时土壤中也存在对其生命具有威胁的各种病原微生物。因此,线虫如何寻找食物并远离病原菌及其它不利因素的威胁,这对其生存具有极其重要的意义。线虫具备较为完善的化感体系,能够通过这些化感神经元检测环境中包括挥发性和水溶性等多种化学物质。而嗅觉信号系统则是其化学感知中的最高效系统之一,嗅觉系统能够检测、感知环境中的挥发性信号,并将这些信号和食物或者危机等环境因素联系在一起,从而判别环境的利害关系,产生趋化或逃避等一系列应激行为,这也是线虫分辨食物与危险的最迅速的工具之一。我们前期的研究已证明：杀线虫芽孢杆菌(Bacillus nematocida) B16菌株拥有很强的侵染线虫的能力,并采用GC-MS鉴定了该菌吸引线虫的挥发性信号分子。在此基础上,本研究通过检测这些挥发性化合物对秀丽隐杆线虫的吸引力,筛选到吲哚和2-乙基己醇这两种具有显著趋化性的物质,并对其信号转导通路进行了确认,从一个方向阐明了细菌与线虫互作的分子机理。本文的主要研究结果包括： 1、验证了吲哚和2-乙基己醇两种吸引线虫的物质,并确定线虫感知它们的主要神经元。对线虫趋化性嗅觉神经元AWA和AWC功能缺失型突变株odr-1、odr-7的趋化性检测,确定吲哚主要通过神经元AWC来吸引线虫；2-乙基己醇主要通过神经元AWA来吸引线虫。 2、证实STR-193为感知吲哚的G蛋白偶联受体；STR-7为感知2-乙基己醇的G蛋白偶联受体。目前在秀丽隐杆线虫的嗅觉研究领域中,对其配体-受体对应关系的报道较少,唯有丁二酮-ODR-10这个在AWA中表达的配受体关系解析较为清楚。通过检测线虫对吲哚和2-乙基己醇这两种物质趋化能力的变化,我们发现str-193RNAi后线虫感知吲哚的能力明显下降,但感知其他AWC神经元感知的趋化物质,如苯甲醛的能力没有变化。而另一方面str-7RNAi的线虫感知2-乙基己醇的能力明显降低,但感知其他AWA神经元感知的趋化物质,如丁二酮也未受影响。从表型来看,STR-193为线虫感知吲哚的特异性受体；STR-7为线虫感知2-乙基己醇的特异性受体。 3、确定G蛋白偶联受体STR-193和STR-7所定位的神经元秀丽隐杆线虫中有大约1000多个G蛋白偶联受体(GPCRs)其中大部分为化感受体,目前绝大多数化感受体没有确定所表达神经元(包括STR-193和STR-7)。本实验通过将带有这两个基因启动子的载体显微注射进入线虫体内进行荧光表达,确定其表达的神经元。通过显微注射确定str-193位于AWC神经元。 4、确定了线虫感知吲哚和2-乙基己醇的嗅觉信号转导途径中其他的蛋白。通过对RNAi和基因突变体检测线虫趋化性改变,发现影响线虫对吲哚识别的基因包含：G蛋白的α亚基(odr-3,gsa-1),鸟苷酸环化酶(odr-1.daf-11),环化核苷酸门控的离子通道亚基(tax-2、tax-4)等成分。测试影响线虫对2-乙基己醇趋化感知的基因,包含：G蛋白的α亚基(odr-3、gpa-6),瞬时感受电位离子通道(ocr-2、osm-9),磷脂酶C(egl-8、plc-])等成分。发现在线虫感知吲哚的过程中,cGMP途径起主要作用；在线虫感知2-乙基己醇的过程当中,PLC途径和TRPV通道起主要作用。本文的创新之处： 1、首次确定了STR-193为吲哚的特异性受体,确定STR-7为2-乙基己醇的特异性受体。 2、首次确定参与吲哚和2-乙基己醇的嗅觉信号传导通路中的相关蛋白。
[Abstract]:Pattern organisms live in the complex soil environment, which feed on the bacteria in the soil, and there are various pathogenic microbes that threaten their lives. Therefore, it is of great significance for the nematodes to find food and to keep away from the threat of pathogens and other adverse factors. The insect has a more perfect allelic system that can detect a variety of chemicals, including volatile and water-soluble substances in the environment, and the olfactory signal system is one of the most efficient systems in its chemical perception. The olfactory system can detect and perceive volatile signals in the environment, and the signals and food or danger. It is also one of the fastest tools of nematodes to distinguish food and risk.
Our previous studies have shown that Bacillus nematocida B16 has a strong ability to infect nematodes and uses GC-MS to identify the volatile signal molecules that attract nematodes. On this basis, this study screened the indole by detecting the attractiveness of these volatile compounds to the Caenorhabditis elegans. And 2- ethylhexyl alcohol, two substances with significant chemotaxis, confirm the signal transduction pathway and elucidate the molecular mechanism of the interaction between bacteria and nematodes from one direction.
The main results of this paper include:
1, two kinds of nematodes were identified by indole and 2- ethylhexanol, and the main neurons were identified by nematodes.
The chemotaxis of the nematode chemotactic neuron AWA and AWC functional deletion mutant odr-1 and ODR-7 was detected, and the indole was determined to attract the nematode mainly through the neuron AWC, and 2- ethyl hexanol mainly attracted the nematode through the neuronal AWA.
2, we confirmed that STR-193 is a G protein coupled receptor that perceiving indole and STR-7 is a G protein coupled receptor that perceiving 2- ethylhexanol.
In the field of olfactory study of Caenorhabditis elegans, there are few reports on its ligand receptor correspondence, only two ketone -ODR-10, which is expressed in AWA, is well resolved. By detecting the changes in the chemotaxis of the two substances of indole and 2- ethylhexanol, we found the perception of the nematode after str-193RNAi. The capacity of indole decreased significantly, but the ability to perceive chemotaxis, such as benzaldehyde, by other AWC neurons did not change. On the other hand, the ability of str-7RNAi to perceive 2- ethylhexanol significantly decreased, but the chemotactic substances perceived by other AWA neurons, such as D two ketone, were also unaffected. From the phenotype, STR-193 was the sense of nematode. STR-7 is a specific receptor for 2- ethylhexanol.
3, identify the neurons localized in the G protein coupled receptors STR-193 and STR-7.
There are about more than 1000 G protein coupled receptors (GPCRs) in the Caenorhabditis elegans, most of which are Allelochemical receptors. Most allelopathic receptors are currently not determined to express neurons (including STR-193 and STR-7). This experiment was made by microinjection of carriers with these two gene promoters into the nematode for fluorescence expression and determined the table Str-193 was located in AWC neurons by microinjection.
4, the other proteins in the olfactory signal transduction pathway of indole and 2- ethyl hexanol were identified.
By measuring the chemotaxis of RNAi and gene mutations, we found that the genes that affect the indole recognition of the nematode include: G protein alpha subunit (odr-3, gsa-1), guanosine cyclase (odr-1.daf-11), cyclized nucleotide gated ion channel subunit (tax-2, tax-4), etc., to test the genes that affect the chemotaxis of 2- ethylhexyl alcohol by nematodes. It includes: odr-3 (gpa-6) of G protein, ocr-2 (osm-9) and phospholipase C (egl-8, plc-]). It is found that the cGMP pathway plays a major role in the process of perceiving indoles by on-line worms, and during the process of perceiving 2- ethylhexanol, the PLC pathway and TRPV channel play a major role in the process of sensing 2- ethylhexanol.
The innovation of this article:
1, for the first time, STR-193 was identified as a specific receptor for indole, and STR-7 was identified as a specific receptor for 2- ethylhexanol.
2, for the first time, the proteins involved in the olfactory signal transduction pathway of indole and 2- ethyl hexanol were first identified.
【学位授予单位】：云南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S154.386

【参考文献】