硼抑制烟草疫霉产孢的相关机制及该菌csn4基因的转录特征

发布时间：2018-02-20 15:03

本文关键词： 烟草疫霉菌矿质元素硼抗氧化系统 COP9信号复合体 csn4基因　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：烟草疫霉(Phytophthora nicotianae)是卵菌纲的一种土传植物病原菌,广泛存在于温带、亚热带和热带,可侵染包括烟草、番茄、柑橘、杏等90多个科250多个属的1000多种植物。烟草疫霉破坏性极强,由其引起的病害严重危害着作物的生长和生理,给农业生产造成了巨大经济损失,因而是一种备受关注的土传植物病原菌。然而,目前尚无有效的方法来控制这种土传病害的传播。研究发现烟草疫霉产孢子囊和释放游动孢子是导致寄主病害发生与流行的主要原因。因此加强烟草疫霉孢子囊产生及游动孢子释放机制的研究,减少和控制烟草疫霉产生孢子囊,对防控烟草疫霉病害发生具有重要意义。本实验室前期研究发现,0.5 mM硼极显著抑制烟草疫霉孢子囊的产生,并发现硼在抑制烟草疫霉产孢子囊时显著抑制了一个与致病疫霉基因组测序推导的COP9信号复合体CSN4亚基同源的基因表达,然而其抑制机理并不清楚。为此本文分析研究了烟草疫霉抗氧化系统和csn4基因对硼抑制的响应,以及csn4基因在烟草疫霉不同生长发育时期的转录特征,结果表明硼显著改变了烟草疫霉的抗氧化系统,csn4基因转录量在烟草疫霉产孢子囊时期显著高于其它时期,添加硼处理会显著抑制烟草疫霉产孢子囊时期csn4基因的转录。研究结果证明了COP9信号复合体CSN4亚基与烟草疫霉产孢子囊有关,为控制烟草疫霉孢子囊的产生提供理论基础。主要取得了如下研究结果:(1)硼对烟草疫霉产孢期抗氧化系统影响的研究结果表明,硼处理组烟草疫霉产孢期的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性显著(p0.05)低于无硼处理,相反,所有硼处理组均使得丙二醛(MDA)含量增加,而MDA作为细胞氧化的终产物,其含量的升高说明活性氧积累,细胞处于氧化应激状态。综合分析表明硼胁迫使得菌体抗氧化酶活性降低,导致细胞处于氧化应激状态,而不能正常发育。(2)通过对引物等的优化,本实验建立了csn4基因的相对定量方法。采用beta-tubulin基因作为内参基因,分别对两基因的引物进行设计并筛选,最终各获得一对特异性良好的引物,并分别构建了两基因的重组质粒,最终建立了csn4和beta-tubulin基因的实时荧光定量标准曲线,其回归方程分别为y=-4.007x+26.889、y=-4.109x+28.453,相关系数R2分别为0.9976、0.9936。采用Ecsn4/Etub的值(Ecsn4和Etub分别表示csn4和beta-tubulin基因的转录量)来表示csn4基因的转录水平,此方法有效的消除了不同处理样品模板量差异对实验结果的影响,并且本研究证明了该定量方法的可行性。(3)不同浓度硼处理下烟草疫霉产孢期csn4基因转录的定量分析结果表明,硼处理组烟草疫霉产孢期csn4基因转录水平显著低于无硼处理,且低浓度的硼处理(0.5 mM)就可显著抑制csn4基因的转录,其抑制率达到40%左右,随硼浓度的变化,csn4基因转录水平差异不显著。(4)对烟草疫霉不同时期csn4基因转录的定量分析结果显示,烟草疫霉从菌丝生长阶段到产孢阶段,csn4基因的转录水平显著升高,且诱导产孢阶段以及游动孢子释放阶段csn4基因的转录水平均显著高于菌丝生长及游动孢子阶段。烟草疫霉csn4基因不同时期转录水平大小顺序为:产孢阶段游动孢子释放菌丝生长游动孢子阶段。在诱导产孢阶段中,随诱导产孢时间的延长,csn4基因的转录水平升高,当诱导12 h时达到最高水平,相当于菌丝生长阶段csn4基因转录量的4倍左右。综合以上结果表明,csn4基因参与烟草疫霉的整个生命过程,但主要参与烟草疫霉的发育产孢阶段,且在诱导产孢12 h时可能是孢子囊物质大量合成的阶段。
[Abstract]:Phytophthoraparasiticavar (Phytophthora nicotianae) is a kind of soil borne oomycete plant pathogen, widespread in temperate, subtropical and tropical, which can infect tobacco, tomato, citrus, apricots and other more than 90 families and more than 250 genera. 1000 species of Phytophthora nicotianae is destructive, caused by the disease serious harm to crop growth and physiology, causing huge economic losses to agricultural production, so it is a concern of the soil borne plant pathogens. However, there is no effective communication method to control the soil borne diseases. The study found that Phytophthora parasitica spore ascus and zoospore release is a major cause of host the disease incidence and prevalence. So the study of tobacco Phytophthora sporangium formation and zoospore release mechanism, reduce and control the tobacco Phytophthora sporangium, is important for the prevention and control of tobacco Phytophthora diseases. The The preliminary study found that 0.5 mM boron significantly restrained tobacco Phytophthora sporangium, and found that boron in inhibiting tobacco Phytophthora sporulation was significantly inhibited with a Phytophthora infestans genome sequencing is the signal of COP9 subunit CSN4 homologous gene expression, but its inhibition mechanism is not clear. Therefore this paper studies the phytophthoraparasiticavar antioxidant system and csn4 gene response to boron inhibition, and csn4 gene transcription in characteristics of Phytophthora nicotianae in different periods of growth. The results show that boron has a significant change in the antioxidant system of Phytophthora parasitica, csn4 gene expressed in tobacco Phytophthora sporulation period was significantly higher than that in other periods, add boron treatment could significantly inhibit the transcription phytophthoraparasiticavar sporulation period csn4 gene. The research results demonstrate that COP9 signaling complex CSN4 subunit related to phytophthoraparasiticavar sporulation, for the control of smoke To provide a theoretical basis to produce grass Phytophthora sporangium. The main results obtained are as follows: (1) study on the effects of boron phytophthoraparasiticavar sporulation phase antioxidant system showed that boron treatment of superoxide dismutase group phytophthoraparasiticavar sporulation stage (SOD), catalase (CAT) activity was significantly (P0.05) is lower than that of boron free treatment, on the contrary, all treatment groups were made of boron malondialdehyde (MDA) content increased, and MDA as the end product of cellular oxidation, the content that increases ROS accumulation and oxidative stress in the cells. The comprehensive analysis indicated that boron stress reduced cell antioxidant activity, leading to oxidative stress in cells state, and not the normal development. (2) through the optimization of primers, this experiment established the relative quantitative method of csn4 gene. The beta-tubulin gene as a reference gene, respectively on the two gene design and screening, the final Get a pair of specific primers were good, and to construct recombinant plasmid two gene, the eventual establishment of real time fluorescence quantitative standard curve of csn4 and beta-tubulin genes, the regression equations were y=-4.007x+26.889, y=-4.109x+28.453, correlation coefficient R2 was 0.9976,0.9936. with the value of Ecsn4/Etub (Ecsn4 and Etub respectively, the amount of csn4 and beta-tubulin gene transcription the) to express the transcription level of csn4 gene, this method effectively eliminates the influence of different sample template volume differences on the experimental results, and this study proved the feasibility of the quantitative method. (3) different concentrations of boron treatment results of quantitative analysis of tobacco Phytophthora sporulation stage transcription of csn4 gene showed that tobacco group Phytophthora sporulation stage csn4 gene transcription level was significantly lower than the treatment of boron boron free treatment, and low concentration of boron (0.5 mM) can significantly inhibit the transcription of csn4 gene, The inhibition rate reached about 40%, with the changes of the boron concentration, the difference of csn4 gene transcription level was not significant. (4) the results of quantitative analysis of Phytophthora nicotianae in different periods of transcription of csn4 gene showed that Phytophthora parasitica from mycelial growth phase to sporulation stage, the transcription level of csn4 gene was significantly increased, and the induction of transcription spore stage and zoospore release stage csn4 gene were significantly higher than that of mycelial growth and zoospore stage. Tobacco Phytophthora csn4 gene transcription level in different periods of the order of sporulation stage zoospore release of zoospores of mycelial growth stage. In the induction of sporulation stage, with the increase of the induced sporulation time, increased transcription level the csn4 gene, reached the highest level when induced by 12 h, equivalent to the mycelial growth stage csn4 gene transcription was about 4 times. The above results showed that the csn4 gene in tobacco Phytophthora whole life The process is mainly involved in the development of the sporulation stage of Phytophthora tobacco, and it may be a stage of mass synthesis of sporovesicles when the spore production of 12 h is induced.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S432.4

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