【摘要】：高粱靶斑病是由絲狀真菌--平臍蠕孢菌(Bipolaris sorghicola)引起的一種高粱葉部真菌病害。該類病原菌能夠產(chǎn)生二倍半萜類毒素--蛇胞菌素(ophiobolin),且該毒素來源于經(jīng)典的MVA(甲羥戊酸)途徑。本研究擬探討蛇孢菌素在高粱靶斑病菌致病過程中的作用及不同條件下毒素合成關(guān)鍵酶編碼基因的表達(dá)情況。首先在不同寄主表皮細(xì)胞中對比觀察該病原菌的侵染特性,后采用MVA途徑的抑制劑--洛伐他汀處理高粱靶斑病菌孢子懸浮液,將其接種于離體高粱葉片上觀察病原菌致病性變化,并與創(chuàng)傷接種和人為添加毒素的病原菌致病性進(jìn)行對比。后采用高效液相色譜-串聯(lián)質(zhì)譜法(LC-MS/MS)測定該病原菌在PDB液體培養(yǎng)基中所產(chǎn)毒素的種類和含量及洛伐他汀處理對其含量的影響;并結(jié)合蛇孢菌素處理后高粱葉片保護(hù)酶活性及質(zhì)膜過氧化程度的變化,綜合分析毒素在病原菌致病過程中的作用。同時(shí),利用qRT-PCR技術(shù)對不同條件下該病原菌毒素合成關(guān)鍵酶編碼基因(IPPI/FPPS/HMGR/GGPPS)的表達(dá)進(jìn)行了檢測。實(shí)驗(yàn)結(jié)果如下:1.高粱靶斑病菌可侵染多種植物。除了高粱外,單子葉植物(如大麥)和雙子葉植物(如洋蔥、擬南芥)均是該病原菌的侵染對象。其在四種植物表皮細(xì)胞中的侵染情況大致相同,均可從細(xì)胞表面和細(xì)胞間隙兩個(gè)途徑進(jìn)入細(xì)胞內(nèi)部。2.高粱靶斑病菌所產(chǎn)毒素以蛇孢菌素A為主。洛伐他汀處理可明顯降低毒素含量,抑制高粱靶斑病菌菌絲在高粱葉表皮細(xì)胞中的侵染,導(dǎo)致病原菌的致病性顯著下降。且在0-1500μg/ml濃度范圍內(nèi),隨著濃度的加大,病原菌致病性的抑制程度也隨之增大,最佳處理時(shí)間和最佳處理濃度分別為24h和1000μg/ml。且人為添加蛇孢菌素和創(chuàng)傷接種均可恢復(fù)病原菌的致病性。此外該毒素可引起高粱葉組織細(xì)胞膜透性改變,膜脂過氧化加劇,MDA含量上升,且隨著毒素處濃度的加大和處理時(shí)間的延長細(xì)胞膜透性和MDA含量持續(xù)增大。同時(shí)還可造成高粱葉片SOD酶活性下降,CAT、APX的酶活性升高。說明蛇孢菌素可影響高粱葉組織細(xì)胞膜透性,造成膜功能的紊亂,同時(shí)影響寄主保護(hù)酶活,使病原菌更易入侵植物。3.IPPI、FPPS、GGPPS和HGMR是高粱靶斑病菌毒素合成過程中的四個(gè)關(guān)鍵酶。我們通過Real Time PCR技術(shù),檢測了高粱靶斑病菌菌絲階段這四個(gè)酶的編碼基因(IPPI、FPPS、GGPPS、HGMR)在不同條件下的基因表達(dá)情況。結(jié)果顯示:不同濃度洛伐他汀處理(25μM、50μM、100μM、200μM)均可導(dǎo)致毒素合成酶編碼基因表達(dá)的下降,且隨著處理濃度的加大抑制程度也隨之增大。而相同濃度的洛伐他汀(100μM)在不同處理時(shí)間(2h、8h、24h、36h、48h)條件下,隨著處理時(shí)間的推移,除了FPPS在8h和36h有反饋性提高外,高粱靶斑病菌菌絲階段毒素合成酶編碼基因IPPI、HMGR和GGPPS的表達(dá)在任一時(shí)間均明顯下降。此外:我們發(fā)現(xiàn)不同溫度、光照、PH條件下IPPI、FPPS、GGPPS表達(dá)模式相似,適當(dāng)?shù)母邷?32℃)均可提高這三種基因的表達(dá);HMGR的表達(dá)模式則與其相反,高溫(32℃)反而不利于該基因的表達(dá),PH4.0的弱酸環(huán)境則促進(jìn)其表達(dá),相比對照組升高近20倍,且HMGR基因?qū)λ嵝原h(huán)境非常敏感。
[Abstract]:Sorghum target spot disease is caused by filamentous fungus Bipolaris sorghicola. This pathogen can produce double sesquiterpene toxin, ophiobolin, and this toxin is derived from the classical MVA pathway. This study is to explore the pathogenesis of ophiobolin in Sorghum target spot pathogen. The infection characteristics of the pathogen were observed in different host epidermal cells. Lovastatin, an inhibitor of MVA pathway, was used to treat the spore suspension of sorghum target spot pathogen and inoculated on Sorghum leaves in vitro to observe the pathogenicity. The pathogenicity of the pathogenic bacteria was compared with that of wound inoculation and artificial addition of toxins. The toxins produced by the pathogen in PDB liquid medium were determined by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the effects of lovastatin treatment on the content of the toxins were also studied. At the same time, the expression of IPPI / FPPS / HMGR / GGPPS was detected by qRT-PCR under different conditions. The results were as follows: 1. Sorghum target spot pathogen can infect many plants. In addition to sorghum, monocotyledons (such as barley) and dicotyledons (such as onions, Arabidopsis) are infected by the pathogen. The infection of the pathogen in the epidermal cells of the four plants is similar, and it can enter the inner cells from the cell surface and intercellular space. 2. Lovastatin treatment can significantly reduce the toxin content, inhibit the infection of sorghum target spot pathogen hyphae in Sorghum leaf epidermal cells, resulting in a significant decrease in pathogenicity of pathogenic bacteria. The pathogenicity of pathogenic bacteria could be restored by adding anthropogenic serotonin and wound inoculation, respectively, 24 h and 1000 ug/ml. In addition, the toxin could cause membrane permeability change, membrane lipid peroxidation and MDA content increase in Sorghum leaf tissue, and cell membrane permeability and MDA content increased with the increase of toxin concentration and treatment time. The results showed that ophiocin could affect the membrane permeability of sorghum leaf tissue, cause membrane dysfunction, affect the activity of host protective enzymes, and make pathogens more likely to invade plants. 3. IPPI, FPPS, GGPPS and HGMR are the four key factors in the process of toxin synthesis of sorghum target spot pathogen. The gene expression of the four enzymes (IPPI, FPPS, GGPPS, HGMR) in the mycelial stage of Sorghum target spot pathogen was detected by Real Time PCR under different conditions. However, the expression of IPPI, HMGR and GGPPS in the mycelial phase of Sorghum target spot pathogen increased with the increase of the concentration of lovastatin (100 mu M) and the treatment time (2 h, 8 h, 24 h, 36 h, 48 h), except for the feedback of FPPS at 8 h and 36 h. In addition, we found that IPPI, FPPS and GGPPS expression patterns were similar at different temperatures, illumination and PH conditions, and the expression of these three genes could be increased at appropriate high temperatures (32 degrees Celsius); on the contrary, high temperatures (32 degrees Celsius) were not conducive to the expression of HMGR gene, and weak acid environment of PH4.0 promoted its expression. The control group increased by nearly 20 times, and the HMGR gene was very sensitive to acidic environment.
【學(xué)位授予單位】：河南師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.14

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 殷如;洪葵;;絲狀真菌二倍半萜化合物及其合成酶[J];生物工程學(xué)報(bào);2016年12期

2 魏潔書;王煥;戴慧明;卿志浩;楊錦芬;;洛伐他丁和膦胺霉素對煙草HMGR和DXR活性的影響[J];農(nóng)業(yè)與技術(shù);2015年03期

3 彭陳;王瑞;陳洪亮;王俊偉;袁藝;郭士偉;;水稻胡麻葉斑病病原菌的培養(yǎng)特性[J];江蘇農(nóng)業(yè)學(xué)報(bào);2014年03期

4 錢延春;劉麗;曹豪杰;吳明;莊園;曲慶玲;趙壽經(jīng);;MVA與MEP途徑抑制劑對人參皂苷合成能力的影響研究[J];特產(chǎn)研究;2013年03期

5 魏潔書;楊錦芬;凌敏;劉卉;詹若挺;陳蔚文;;茉莉酸甲酯調(diào)控陽春砂HMGR、DXR和DXS基因表達(dá)[J];廣州中醫(yī)藥大學(xué)學(xué)報(bào);2013年01期

6 陳洪亮;彭陳;王俊偉;袁藝;郭士偉;;水稻胡麻葉斑病病原菌的分離及鑒定[J];西北農(nóng)林科技大學(xué)學(xué)報(bào)(自然科學(xué)版);2012年08期

7 程亮;朱海霞;郭青云;;鐮孢菌毒素對野燕麥根尖組織防御酶活性的影響[J];江蘇農(nóng)業(yè)科學(xué);2012年07期

8 張園園;徐秀德;王振東;張立軍;姜鈺;胡蘭;董懷玉;;高粱靶斑病菌粗毒素對高粱胚根生長及胚根細(xì)胞膜透性的影響[J];中國農(nóng)學(xué)通報(bào);2012年12期

9 張園園;徐秀德;徐婧;劉可杰;姜鈺;胡蘭;;高粱靶斑病病原菌種群多樣性研究[J];沈陽農(nóng)業(yè)大學(xué)學(xué)報(bào);2012年02期

10 李志勇;賈麗霞;董金皋;董志平;;玉米小斑病菌鈣調(diào)素基因的克隆與序列分析[J];江蘇農(nóng)業(yè)學(xué)報(bào);2011年05期

相關(guān)博士學(xué)位論文 前3條

1 任昂;茉莉酸甲酯對靈芝三萜生物合成的影響及其靈芝應(yīng)答基因的差異表達(dá)研究[D];南京農(nóng)業(yè)大學(xué);2012年

2 張園園;高粱靶斑病菌致病機(jī)理及抗病基因分子標(biāo)記定位[D];沈陽農(nóng)業(yè)大學(xué);2012年

3 臺(tái)蓮梅;馬鈴薯早疫病菌多樣性和侵染過程及品種抗病機(jī)制研究[D];黑龍江八一農(nóng)墾大學(xué);2011年

相關(guān)碩士學(xué)位論文 前2條

1 賈蕊鴻;馬鈴薯立枯絲核菌毒素對植株細(xì)胞膜傷害機(jī)理的研究[D];甘肅農(nóng)業(yè)大學(xué);2016年

2 劉麗;利用抑制劑對人參皂苷生物合成途徑MVA與MEP的研究[D];吉林大學(xué);2012年

，

本文編號(hào)：2238327