本文選題：丁香假單胞菌 + 轉(zhuǎn)座突變文庫�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：目前世界上已發(fā)現(xiàn)超過4000種植物寄生線蟲,約占所有線蟲的10%。植物寄生線蟲主要寄生于植物根、莖、葉等部位上,通過口針刺入細(xì)胞組織吸取植物營養(yǎng)物質(zhì)。世界上每年因植物寄生線蟲造成的農(nóng)林業(yè)經(jīng)濟(jì)損失已達(dá)上千億。傳統(tǒng)的防治線蟲方法主要有物理和化學(xué)防治法,其中最行之有效的方法是化學(xué)農(nóng)藥法。但這些化學(xué)殺蟲劑難以降解,會(huì)對(duì)環(huán)境造成不可逆的破壞,而且農(nóng)藥殘留在農(nóng)作物上也會(huì)對(duì)人類造成危害。因此對(duì)環(huán)境無害具有較好防治效果的生物防治方法漸漸成為人們的研究熱點(diǎn)。目前主要生物防治微生物有食線蟲真菌(fungi)、蘇云金芽孢桿菌(Bacillus thuring-iensis)、銅綠假單胞菌(Pseudomonas aeruginosa)等。丁香假單胞菌(Pseudomonas syringae)MB03是本實(shí)驗(yàn)室從凍害植物組織上分離出的一株對(duì)秀麗隱桿線蟲(Caenorhabditis elegans)及南方根結(jié)線蟲(Meloidogyne incongnita)均具有較高毒性的菌株。丁香假單胞菌是一種植物致病菌,能產(chǎn)生冰核蛋白使植物發(fā)生凍害,但迄今為止還很少有關(guān)于它對(duì)動(dòng)物具有致病性報(bào)道。銅綠假單胞菌對(duì)秀麗隱桿線蟲致病研究目前已非常深入,眾多的重要毒性基因已經(jīng)被發(fā)現(xiàn),但關(guān)于銅綠假單胞菌中毒性物質(zhì)對(duì)南方根結(jié)線蟲作用卻少有報(bào)道。為了鑒定丁香假單胞菌MB03中對(duì)線蟲毒性基因并研究它們致病機(jī)制,本實(shí)驗(yàn)室利用了pUT mini-Tn5Km2轉(zhuǎn)座子構(gòu)建了包括1256個(gè)突變株的轉(zhuǎn)座突變文庫并利用液體殺蟲的方法從中篩選出12株對(duì)線蟲毒性衰弱菌株。通過hiTAIL-PCR(熱不對(duì)稱PCR)方法鑒定出7個(gè)突變株對(duì)應(yīng)轉(zhuǎn)座突變基因。為了驗(yàn)證這些基因在對(duì)線蟲毒性中起到的作用,我們構(gòu)建了其中6個(gè)基因重組質(zhì)粒并進(jìn)行相關(guān)蛋白的誘導(dǎo)和純化。一共有5個(gè)蛋白被純化出來并進(jìn)行了下一步秀麗隱桿線蟲相關(guān)生測(cè)實(shí)驗(yàn)(致死率、產(chǎn)卵率、運(yùn)動(dòng)性、生長抑制等)及南方根結(jié)線蟲毒殺實(shí)驗(yàn)。實(shí)驗(yàn)結(jié)果表明VT47_06935,zapE,oprD對(duì)秀麗隱桿線蟲具有一定毒性,它們LC_(50)值分別為259.7μg/ml,268.5μg/ml,147.3μg/ml。另外對(duì)線蟲產(chǎn)卵率影響方面,zapE,VT47_06935以及VT47_19645蛋白在濃度為2_(50)μg/ml時(shí)分別對(duì)線蟲產(chǎn)卵率減少了18%,32.4%,14%。線蟲運(yùn)動(dòng)性影響結(jié)果表明在蛋白濃度為200μg/ml時(shí),VT47_06935,zapE以及VT47_19645蛋白作用線蟲后運(yùn)動(dòng)振幅分別減少了29%,22%,14%。另外熒光觀察發(fā)現(xiàn)VT47_06935蛋白分布與線蟲全身,推測(cè)其可能破壞線蟲腸道最終引起線蟲死亡,而zapE和VT47_19645則主要分布與線蟲腸道內(nèi),說明其并不是通過破壞腸道引起線蟲死亡,具體的作用機(jī)制還需進(jìn)一步實(shí)驗(yàn)證實(shí)。最后利用這幾個(gè)蛋白對(duì)于南方根結(jié)線蟲殺蟲實(shí)驗(yàn),遺憾的是只有zapE在濃度為2_(50)μg/ml時(shí)第7天致死率有38.5%,VT47_19645和VT47_06935則完全沒有毒性。針對(duì)銅綠假單胞菌,本研究以銅綠假單胞菌ATCC 15442及CMCC(B)10104為研究對(duì)象,篩選出其中可能起到直接殺線蟲的毒性基因,并進(jìn)行重組質(zhì)粒的構(gòu)建,總計(jì)構(gòu)建了11個(gè)相關(guān)基因重組質(zhì)粒并對(duì)其進(jìn)行了蛋白的誘導(dǎo)純化。其中共有6個(gè)菌株誘導(dǎo)出目的蛋白,分別為lasB、exoS、clpA、clpP、plcH、pepP,而且除lasB基因外都純化出相應(yīng)目的蛋白。對(duì)已純化出的目的蛋白進(jìn)行南方根結(jié)線蟲殺蟲實(shí)驗(yàn)。結(jié)果表明exoS、clpA對(duì)南方根結(jié)線蟲毒性較弱,在濃度為200μg/ml時(shí)5天致死率分別為30.2%和34.2%,而pepP蛋白毒性相對(duì)較高,同樣條件下具有約60.1%致死率。
[Abstract]:At present, more than 4000 species of parasitic nematodes have been found in the world. The 10%. parasitic nematodes that account for all the nematodes are mainly parasitic on the plant roots, stems, leaves and other parts of the plant. The plant nutrients are absorbed by the mouth needle into the cell tissue. The economic loss of Agroforestry caused by plant parasitic nematodes has reached hundreds of billions of years in the world. The most effective methods are chemical and chemical control methods, and the most effective method is chemical pesticide. However, these chemical pesticides are difficult to degrade, cause irreversible damage to the environment, and the pesticide residues will cause harm to mankind. Therefore, the biological control method of good prevention and control effect on the environment is gradual. At present, the main biological control microorganisms are nematode fungi (fungi), Bacillus thuringiensis (Bacillus thuring-iensis), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and so on. The Pseudomonas Syringa (Pseudomonas syringae) MB03 is a pair of beautiful hidden lines isolated from the frozen plant tissue. Caenorhabditis elegans and the southern root knot nematode (Meloidogyne incongnita) all have a highly toxic strain. Pseudomonas Syringa is a plant pathogenic bacteria that produces ice nucleation proteins that cause frost damage to plants, but so far there are few reports on its pathogenicity to animals. Pseudomonas aeruginosa has been caused by Pseudomonas aeruginosa to the Caenorhabditis elegans. Many important toxic genes have been found, but there are few reports on the effect of Pseudomonas aeruginosa toxic substances on the southern root knot nematode. In order to identify the toxic genes in the Pseudomonas Syringa MB03 and to study the pathogenesis of them, the laboratory used the pUT mini-Tn5Km2 transposon. 12 strains of nematode virulence strains were screened from the transposable mutant library of 1256 mutant strains, and 7 mutant strains were identified by the method of hiTAIL-PCR (thermal asymmetric PCR). In order to verify the effect of these genes on the toxicity of nematodes, we constructed 6 of them. The recombinant plasmids were induced and purified. A total of 5 proteins were purified and carried out the next test for the next step of Caenorhabditis elegans (lethality, oviposition rate, motility, growth inhibition, etc.) and the southern root knot nematode toxicity test. The results showed that VT47_06935, zapE and oprD had certain toxicity to the Caenorhabditis elegans. Their LC_ (50) values were 259.7 g/ml, 268.5 mu g/ml and 147.3 mu g/ml. respectively, and zapE, VT47_06935, and VT47_19645 protein decreased 18%, 32.4%, and 14%. nematodes at the concentration of 2_ (50) micron, respectively, and showed that when the protein concentration was 200 mu g/ml, VT47_06935, VT47_06935, and 14%. were found. The motion amplitude of T47_19645 protein was reduced by 29%, 22%, respectively. The distribution of VT47_06935 protein and the whole body of nematode were observed by 14%.. It was presumed that it might destroy the nematode gut and eventually cause the death of nematode, while zapE and VT47_19645 were mainly distributed in the intestinal tract of the nematode, indicating that it did not cause the death of the nematode by destroying the intestines. Further experiments were needed to confirm the specific mechanism. Finally, using these proteins for the insecticidal experiment of the southern root knot nematode, it was regrettable that only zapE was 38.5% at the concentration of 2_ (50) mu g/ml, while VT47_19645 and VT47_06935 were completely no toxic. For Pseudomonas aeruginosa, this study was based on Pseudomonas aeruginosa ATCC 15442. And CMCC (B) 10104 as the research object, we screened out the toxic genes of direct nematicids and constructed the recombinant plasmids. A total of 11 recombinant plasmids were constructed and purified. Among them, 6 strains were induced to induce egg white, which were lasB, exoS, clpA, clpP, plcH, pepP, and except las. The purified target protein was purified from the B gene. The purified target protein was killed in the southern root knot nematode. The results showed that exoS and clpA were less toxic to the southern root knot nematode. The mortality rate of the 5 days was 30.2% and 34.2% at the concentration of 200 u g/ml, while the toxicity of pepP protein was relatively high, and the rate of death was about 60.1% under the same condition.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S432.45;S476

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 王炳太;;植物根結(jié)的形成與衰敗[J];現(xiàn)代農(nóng)業(yè)科技;2016年21期

2 李娟;張克勤;;食線蟲微生物防控病原線蟲的研究[J];中國生物防治學(xué)報(bào);2013年04期

3 ;The effect of pmpR on the type Ⅲ secretion system in Pseudomonas aeruginosa[J];Chinese Science Bulletin;2012年19期

4 張穎;李國紅;張克勤;;食線蟲真菌資源研究概況[J];菌物學(xué)報(bào);2011年06期

5 周雨朦;陳代杰;;秀麗隱桿線蟲在藥物篩選中的應(yīng)用[J];上海醫(yī)藥;2011年11期

6 趙晴;蔣nInI;;秀麗隱桿線蟲研究綜述[J];安徽農(nóng)業(yè)科學(xué);2010年19期

7 宋磊;張雪洪;;在銅綠假單胞菌PAO1和PA14中確定基因島的新方法[J];科學(xué)通報(bào);2009年20期

8 余子全;王乾蘭;劉斌;鄒雪;喻子牛;孫明;;蘇云金芽胞桿菌伴胞晶體蛋白對(duì)植物寄生線蟲生物測(cè)定方法的建立和高毒力菌株的篩選[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2007年05期

9 張世清;任文彬;王華;黃俊生;;植物寄生線蟲生物防治和抗線蟲基因工程綜述[J];熱帶農(nóng)業(yè)科學(xué);2006年04期

10 劉青娥;曹鵬飛;;植物線蟲的研究和防治[J];安徽農(nóng)業(yè)科學(xué);2006年18期

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