本文關(guān)鍵詞： 煙草疫霉菌 礦質(zhì)元素硼 抗氧化系統(tǒng) COP9信號(hào)復(fù)合體 csn4基因　出處：《西南大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)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(gè)科250多個(gè)屬的1000多種植物。煙草疫霉破壞性極強(qiáng),由其引起的病害嚴(yán)重危害著作物的生長(zhǎng)和生理,給農(nóng)業(yè)生產(chǎn)造成了巨大經(jīng)濟(jì)損失,因而是一種備受關(guān)注的土傳植物病原菌。然而,目前尚無(wú)有效的方法來(lái)控制這種土傳病害的傳播。研究發(fā)現(xiàn)煙草疫霉產(chǎn)孢子囊和釋放游動(dòng)孢子是導(dǎo)致寄主病害發(fā)生與流行的主要原因。因此加強(qiáng)煙草疫霉孢子囊產(chǎn)生及游動(dòng)孢子釋放機(jī)制的研究,減少和控制煙草疫霉產(chǎn)生孢子囊,對(duì)防控?zé)煵菀呙共『Πl(fā)生具有重要意義。本實(shí)驗(yàn)室前期研究發(fā)現(xiàn),0.5 mM硼極顯著抑制煙草疫霉孢子囊的產(chǎn)生,并發(fā)現(xiàn)硼在抑制煙草疫霉產(chǎn)孢子囊時(shí)顯著抑制了一個(gè)與致病疫霉基因組測(cè)序推導(dǎo)的COP9信號(hào)復(fù)合體CSN4亞基同源的基因表達(dá),然而其抑制機(jī)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?qū)ε鹨种频捻憫?yīng),以及csn4基因在煙草疫霉不同生長(zhǎng)發(fā)育時(shí)期的轉(zhuǎn)錄特征,結(jié)果表明硼顯著改變了煙草疫霉的抗氧化系統(tǒng),csn4基因轉(zhuǎn)錄量在煙草疫霉產(chǎn)孢子囊時(shí)期顯著高于其它時(shí)期,添加硼處理會(huì)顯著抑制煙草疫霉產(chǎn)孢子囊時(shí)期csn4基因的轉(zhuǎn)錄。研究結(jié)果證明了COP9信號(hào)復(fù)合體CSN4亞基與煙草疫霉產(chǎn)孢子囊有關(guān),為控制煙草疫霉孢子囊的產(chǎn)生提供理論基礎(chǔ)。主要取得了如下研究結(jié)果:(1)硼對(duì)煙草疫霉產(chǎn)孢期抗氧化系統(tǒng)影響的研究結(jié)果表明,硼處理組煙草疫霉產(chǎn)孢期的超氧化物歧化酶(SOD)、過(guò)氧化氫酶(CAT)活性顯著(p0.05)低于無(wú)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(xì)胞氧化的終產(chǎn)物,其含量的升高說(shuō)明活性氧積累,細(xì)胞處于氧化應(yīng)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(dǎo)致細(xì)胞處于氧化應(yīng)激狀態(tài),而不能正常發(fā)育。(2)通過(guò)對(duì)引物等的優(yōu)化,本實(shí)驗(yàn)建立了csn4基因的相對(duì)定量方法。采用beta-tubulin基因作為內(nèi)參基因,分別對(duì)兩基因的引物進(jìn)行設(shè)計(jì)并篩選,最終各獲得一對(duì)特異性良好的引物,并分別構(gòu)建了兩基因的重組質(zhì)粒,最終建立了csn4和beta-tubulin基因的實(shí)時(shí)熒光定量標(biāo)準(zhǔn)曲線,其回歸方程分別為y=-4.007x+26.889、y=-4.109x+28.453,相關(guān)系數(shù)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(zhuǎn)錄量)來(lái)表示csn4基因的轉(zhuǎn)錄水平,此方法有效的消除了不同處理樣品模板量差異對(duì)實(shí)驗(yàn)結(jié)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(chǎn)孢期csn4基因轉(zhuǎn)錄的定量分析結(jié)果表明,硼處理組煙草疫霉產(chǎn)孢期csn4基因轉(zhuǎn)錄水平顯著低于無(wú)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(zhuǎn)錄,其抑制率達(dá)到40%左右,隨硼濃度的變化,csn4基因轉(zhuǎn)錄水平差異不顯著。(4)對(duì)煙草疫霉不同時(shí)期csn4基因轉(zhuǎn)錄的定量分析結(jié)果顯示,煙草疫霉從菌絲生長(zhǎng)階段到產(chǎn)孢階段,csn4基因的轉(zhuǎn)錄水平顯著升高,且誘導(dǎo)產(chǎn)孢階段以及游動(dòng)孢子釋放階段csn4基因的轉(zhuǎn)錄水平均顯著高于菌絲生長(zhǎng)及游動(dòng)孢子階段。煙草疫霉csn4基因不同時(shí)期轉(zhuǎn)錄水平大小順序?yàn)?產(chǎn)孢階段游動(dòng)孢子釋放菌絲生長(zhǎng)游動(dòng)孢子階段。在誘導(dǎo)產(chǎn)孢階段中,隨誘導(dǎo)產(chǎn)孢時(shí)間的延長(zhǎng),csn4基因的轉(zhuǎn)錄水平升高,當(dāng)誘導(dǎo)12 h時(shí)達(dá)到最高水平,相當(dāng)于菌絲生長(zhǎng)階段csn4基因轉(zhuǎn)錄量的4倍左右。綜合以上結(jié)果表明,csn4基因參與煙草疫霉的整個(gè)生命過(guò)程,但主要參與煙草疫霉的發(fā)育產(chǎn)孢階段,且在誘導(dǎo)產(chǎn)孢12 h時(shí)可能是孢子囊物質(zhì)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(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S432.4

【參考文獻(xiàn)】

相關(guān)期刊論文 前6條

1 李娜;李振輪;楊興有;楊笑笑;何凱;楊水英;;11種礦質(zhì)營(yíng)養(yǎng)元素對(duì)煙草疫霉菌菌絲生長(zhǎng)的影響[J];江蘇農(nóng)業(yè)科學(xué);2014年07期

2 王晗;嚴(yán)占勇;張定貴;吳衛(wèi)玲;李洪;李振輪;;一種土壤中煙草疫霉菌的快速分離鑒定及計(jì)數(shù)方法[J];中國(guó)農(nóng)學(xué)通報(bào);2012年09期

3 楊軍;田香華;羅朝鵬;奚家勤;魏春陽(yáng);金立鋒;薛超群;宋紀(jì)真;;煙草根際土壤浸出液刺激煙草疫霉產(chǎn)孢和接種研究[J];中國(guó)煙草學(xué)報(bào);2011年02期

4 冀華;王建明;李新鳳;;硼抑制辣椒枯萎病菌作用機(jī)理的初步研究[J];河北農(nóng)業(yè)科學(xué);2011年02期

5 黃芳;王建明;徐玉梅;;硼抑制灰霉病菌孢子萌發(fā)機(jī)制的初步研究[J];植物病理學(xué)報(bào);2008年04期

6 王萬(wàn)能;全學(xué)軍;肖崇剛;;煙草疫霉的產(chǎn)孢和接種方法研究[J];植物保護(hù)學(xué)報(bào);2005年01期

相關(guān)碩士學(xué)位論文 前1條

1 李娜;營(yíng)養(yǎng)元素對(duì)煙草疫霉生長(zhǎng)發(fā)育及硼對(duì)產(chǎn)孢期基因轉(zhuǎn)錄的影響[D];西南大學(xué);2014年

，

本文編號(hào)：1519257