本文選題：趨化性 + 吲哚��； 參考：《云南大學(xué)》2015年碩士論文

【摘要】：模式生物秀麗隱桿線蟲生活在復(fù)雜的土壤環(huán)境中,它以土壤中的細(xì)菌作為食,同時(shí)土壤中也存在對(duì)其生命具有威脅的各種病原微生物。因此,線蟲如何尋找食物并遠(yuǎn)離病原菌及其它不利因素的威脅,這對(duì)其生存具有極其重要的意義。線蟲具備較為完善的化感體系,能夠通過(guò)這些化感神經(jīng)元檢測(cè)環(huán)境中包括揮發(fā)性和水溶性等多種化學(xué)物質(zhì)。而嗅覺信號(hào)系統(tǒng)則是其化學(xué)感知中的最高效系統(tǒng)之一,嗅覺系統(tǒng)能夠檢測(cè)、感知環(huán)境中的揮發(fā)性信號(hào),并將這些信號(hào)和食物或者危機(jī)等環(huán)境因素聯(lián)系在一起,從而判別環(huán)境的利害關(guān)系,產(chǎn)生趨化或逃避等一系列應(yīng)激行為,這也是線蟲分辨食物與危險(xiǎn)的最迅速的工具之一。 我們前期的研究已證明：殺線蟲芽孢桿菌(Bacillus nematocida) B16菌株擁有很強(qiáng)的侵染線蟲的能力,并采用GC-MS鑒定了該菌吸引線蟲的揮發(fā)性信號(hào)分子。在此基礎(chǔ)上,本研究通過(guò)檢測(cè)這些揮發(fā)性化合物對(duì)秀麗隱桿線蟲的吸引力,篩選到吲哚和2-乙基己醇這兩種具有顯著趨化性的物質(zhì),并對(duì)其信號(hào)轉(zhuǎn)導(dǎo)通路進(jìn)行了確認(rèn),從一個(gè)方向闡明了細(xì)菌與線蟲互作的分子機(jī)理。 本文的主要研究結(jié)果包括： 1、驗(yàn)證了吲哚和2-乙基己醇兩種吸引線蟲的物質(zhì),并確定線蟲感知它們的主要神經(jīng)元。 對(duì)線蟲趨化性嗅覺神經(jīng)元AWA和AWC功能缺失型突變株odr-1、odr-7的趨化性檢測(cè),確定吲哚主要通過(guò)神經(jīng)元AWC來(lái)吸引線蟲；2-乙基己醇主要通過(guò)神經(jīng)元AWA來(lái)吸引線蟲。 2、證實(shí)STR-193為感知吲哚的G蛋白偶聯(lián)受體；STR-7為感知2-乙基己醇的G蛋白偶聯(lián)受體。 目前在秀麗隱桿線蟲的嗅覺研究領(lǐng)域中,對(duì)其配體-受體對(duì)應(yīng)關(guān)系的報(bào)道較少,唯有丁二酮-ODR-10這個(gè)在AWA中表達(dá)的配受體關(guān)系解析較為清楚。通過(guò)檢測(cè)線蟲對(duì)吲哚和2-乙基己醇這兩種物質(zhì)趨化能力的變化,我們發(fā)現(xiàn)str-193RNAi后線蟲感知吲哚的能力明顯下降,但感知其他AWC神經(jīng)元感知的趨化物質(zhì),如苯甲醛的能力沒有變化。而另一方面str-7RNAi的線蟲感知2-乙基己醇的能力明顯降低,但感知其他AWA神經(jīng)元感知的趨化物質(zhì),如丁二酮也未受影響。從表型來(lái)看,STR-193為線蟲感知吲哚的特異性受體；STR-7為線蟲感知2-乙基己醇的特異性受體。 3、確定G蛋白偶聯(lián)受體STR-193和STR-7所定位的神經(jīng)元 秀麗隱桿線蟲中有大約1000多個(gè)G蛋白偶聯(lián)受體(GPCRs)其中大部分為化感受體,目前絕大多數(shù)化感受體沒有確定所表達(dá)神經(jīng)元(包括STR-193和STR-7)。本實(shí)驗(yàn)通過(guò)將帶有這兩個(gè)基因啟動(dòng)子的載體顯微注射進(jìn)入線蟲體內(nèi)進(jìn)行熒光表達(dá),確定其表達(dá)的神經(jīng)元。通過(guò)顯微注射確定str-193位于AWC神經(jīng)元。 4、確定了線蟲感知吲哚和2-乙基己醇的嗅覺信號(hào)轉(zhuǎn)導(dǎo)途徑中其他的蛋白。 通過(guò)對(duì)RNAi和基因突變體檢測(cè)線蟲趨化性改變,發(fā)現(xiàn)影響線蟲對(duì)吲哚識(shí)別的基因包含：G蛋白的α亞基(odr-3,gsa-1),鳥苷酸環(huán)化酶(odr-1.daf-11),環(huán)化核苷酸門控的離子通道亞基(tax-2、tax-4)等成分。測(cè)試影響線蟲對(duì)2-乙基己醇趨化感知的基因,包含：G蛋白的α亞基(odr-3、gpa-6),瞬時(shí)感受電位離子通道(ocr-2、osm-9),磷脂酶C(egl-8、plc-])等成分。發(fā)現(xiàn)在線蟲感知吲哚的過(guò)程中,cGMP途徑起主要作用；在線蟲感知2-乙基己醇的過(guò)程當(dāng)中,PLC途徑和TRPV通道起主要作用。 本文的創(chuàng)新之處： 1、首次確定了STR-193為吲哚的特異性受體,確定STR-7為2-乙基己醇的特異性受體。 2、首次確定參與吲哚和2-乙基己醇的嗅覺信號(hào)傳導(dǎo)通路中的相關(guān)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.
【學(xué)位授予單位】：云南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S154.386

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本文編號(hào)：1911148