丁香假单胞菌MB03及铜绿假单胞菌中对线虫毒性蛋白筛选与鉴定

发布时间：2018-05-02 18:15

本文选题：丁香假单胞菌 + 转座突变文库　；参考：《华中农业大学》2017年硕士论文

【摘要】：目前世界上已发现超过4000种植物寄生线虫,约占所有线虫的10%。植物寄生线虫主要寄生于植物根、茎、叶等部位上,通过口针刺入细胞组织吸取植物营养物质。世界上每年因植物寄生线虫造成的农林业经济损失已达上千亿。传统的防治线虫方法主要有物理和化学防治法,其中最行之有效的方法是化学农药法。但这些化学杀虫剂难以降解,会对环境造成不可逆的破坏,而且农药残留在农作物上也会对人类造成危害。因此对环境无害具有较好防治效果的生物防治方法渐渐成为人们的研究热点。目前主要生物防治微生物有食线虫真菌(fungi)、苏云金芽孢杆菌(Bacillus thuring-iensis)、铜绿假单胞菌(Pseudomonas aeruginosa)等。丁香假单胞菌(Pseudomonas syringae)MB03是本实验室从冻害植物组织上分离出的一株对秀丽隐杆线虫(Caenorhabditis elegans)及南方根结线虫(Meloidogyne incongnita)均具有较高毒性的菌株。丁香假单胞菌是一种植物致病菌,能产生冰核蛋白使植物发生冻害,但迄今为止还很少有关于它对动物具有致病性报道。铜绿假单胞菌对秀丽隐杆线虫致病研究目前已非常深入,众多的重要毒性基因已经被发现,但关于铜绿假单胞菌中毒性物质对南方根结线虫作用却少有报道。为了鉴定丁香假单胞菌MB03中对线虫毒性基因并研究它们致病机制,本实验室利用了pUT mini-Tn5Km2转座子构建了包括1256个突变株的转座突变文库并利用液体杀虫的方法从中筛选出12株对线虫毒性衰弱菌株。通过hiTAIL-PCR(热不对称PCR)方法鉴定出7个突变株对应转座突变基因。为了验证这些基因在对线虫毒性中起到的作用,我们构建了其中6个基因重组质粒并进行相关蛋白的诱导和纯化。一共有5个蛋白被纯化出来并进行了下一步秀丽隐杆线虫相关生测实验(致死率、产卵率、运动性、生长抑制等)及南方根结线虫毒杀实验。实验结果表明VT47_06935,zapE,oprD对秀丽隐杆线虫具有一定毒性,它们LC_(50)值分别为259.7μg/ml,268.5μg/ml,147.3μg/ml。另外对线虫产卵率影响方面,zapE,VT47_06935以及VT47_19645蛋白在浓度为2_(50)μg/ml时分别对线虫产卵率减少了18%,32.4%,14%。线虫运动性影响结果表明在蛋白浓度为200μg/ml时,VT47_06935,zapE以及VT47_19645蛋白作用线虫后运动振幅分别减少了29%,22%,14%。另外荧光观察发现VT47_06935蛋白分布与线虫全身,推测其可能破坏线虫肠道最终引起线虫死亡,而zapE和VT47_19645则主要分布与线虫肠道内,说明其并不是通过破坏肠道引起线虫死亡,具体的作用机制还需进一步实验证实。最后利用这几个蛋白对于南方根结线虫杀虫实验,遗憾的是只有zapE在浓度为2_(50)μg/ml时第7天致死率有38.5%,VT47_19645和VT47_06935则完全没有毒性。针对铜绿假单胞菌,本研究以铜绿假单胞菌ATCC 15442及CMCC(B)10104为研究对象,筛选出其中可能起到直接杀线虫的毒性基因,并进行重组质粒的构建,总计构建了11个相关基因重组质粒并对其进行了蛋白的诱导纯化。其中共有6个菌株诱导出目的蛋白,分别为lasB、exoS、clpA、clpP、plcH、pepP,而且除lasB基因外都纯化出相应目的蛋白。对已纯化出的目的蛋白进行南方根结线虫杀虫实验。结果表明exoS、clpA对南方根结线虫毒性较弱,在浓度为200μg/ml时5天致死率分别为30.2%和34.2%,而pepP蛋白毒性相对较高,同样条件下具有约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.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.45;S476

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