本文關鍵詞： 煙草疫霉菌 礦質元素硼 抗氧化系統(tǒng) COP9信號復合體 csn4基因　出處：《西南大學》2017年碩士論文　論文類型：學位論文

【摘要】：煙草疫霉(Phytophthora nicotianae)是卵菌綱的一種土傳植物病原菌,廣泛存在于溫帶、亞熱帶和熱帶,可侵染包括煙草、番茄、柑橘、杏等90多個科250多個屬的1000多種植物。煙草疫霉破壞性極強,由其引起的病害嚴重危害著作物的生長和生理,給農業(yè)生產造成了巨大經濟損失,因而是一種備受關注的土傳植物病原菌。然而,目前尚無有效的方法來控制這種土傳病害的傳播。研究發(fā)現煙草疫霉產孢子囊和釋放游動孢子是導致寄主病害發(fā)生與流行的主要原因。因此加強煙草疫霉孢子囊產生及游動孢子釋放機制的研究,減少和控制煙草疫霉產生孢子囊,對防控煙草疫霉病害發(fā)生具有重要意義。本實驗室前期研究發(fā)現,0.5 mM硼極顯著抑制煙草疫霉孢子囊的產生,并發(fā)現硼在抑制煙草疫霉產孢子囊時顯著抑制了一個與致病疫霉基因組測序推導的COP9信號復合體CSN4亞基同源的基因表達,然而其抑制機理并不清楚。為此本文分析研究了煙草疫霉抗氧化系統(tǒng)和csn4基因對硼抑制的響應,以及csn4基因在煙草疫霉不同生長發(fā)育時期的轉錄特征,結果表明硼顯著改變了煙草疫霉的抗氧化系統(tǒng),csn4基因轉錄量在煙草疫霉產孢子囊時期顯著高于其它時期,添加硼處理會顯著抑制煙草疫霉產孢子囊時期csn4基因的轉錄。研究結果證明了COP9信號復合體CSN4亞基與煙草疫霉產孢子囊有關,為控制煙草疫霉孢子囊的產生提供理論基礎。主要取得了如下研究結果:(1)硼對煙草疫霉產孢期抗氧化系統(tǒng)影響的研究結果表明,硼處理組煙草疫霉產孢期的超氧化物歧化酶(SOD)、過氧化氫酶(CAT)活性顯著(p0.05)低于無硼處理,相反,所有硼處理組均使得丙二醛(MDA)含量增加,而MDA作為細胞氧化的終產物,其含量的升高說明活性氧積累,細胞處于氧化應激狀態(tài)。綜合分析表明硼脅迫使得菌體抗氧化酶活性降低,導致細胞處于氧化應激狀態(tài),而不能正常發(fā)育。(2)通過對引物等的優(yōu)化,本實驗建立了csn4基因的相對定量方法。采用beta-tubulin基因作為內參基因,分別對兩基因的引物進行設計并篩選,最終各獲得一對特異性良好的引物,并分別構建了兩基因的重組質粒,最終建立了csn4和beta-tubulin基因的實時熒光定量標準曲線,其回歸方程分別為y=-4.007x+26.889、y=-4.109x+28.453,相關系數R2分別為0.9976、0.9936。采用Ecsn4/Etub的值(Ecsn4和Etub分別表示csn4和beta-tubulin基因的轉錄量)來表示csn4基因的轉錄水平,此方法有效的消除了不同處理樣品模板量差異對實驗結果的影響,并且本研究證明了該定量方法的可行性。(3)不同濃度硼處理下煙草疫霉產孢期csn4基因轉錄的定量分析結果表明,硼處理組煙草疫霉產孢期csn4基因轉錄水平顯著低于無硼處理,且低濃度的硼處理(0.5 mM)就可顯著抑制csn4基因的轉錄,其抑制率達到40%左右,隨硼濃度的變化,csn4基因轉錄水平差異不顯著。(4)對煙草疫霉不同時期csn4基因轉錄的定量分析結果顯示,煙草疫霉從菌絲生長階段到產孢階段,csn4基因的轉錄水平顯著升高,且誘導產孢階段以及游動孢子釋放階段csn4基因的轉錄水平均顯著高于菌絲生長及游動孢子階段。煙草疫霉csn4基因不同時期轉錄水平大小順序為:產孢階段游動孢子釋放菌絲生長游動孢子階段。在誘導產孢階段中,隨誘導產孢時間的延長,csn4基因的轉錄水平升高,當誘導12 h時達到最高水平,相當于菌絲生長階段csn4基因轉錄量的4倍左右。綜合以上結果表明,csn4基因參與煙草疫霉的整個生命過程,但主要參與煙草疫霉的發(fā)育產孢階段,且在誘導產孢12 h時可能是孢子囊物質大量合成的階段。
[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.

【學位授予單位】：西南大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S432.4

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