【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鍵酶編碼基因的表達情況。首先在不同寄主表皮細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行對比。后采用高效液相色譜-串聯(lián)質譜法(LC-MS/MS)測定該病原菌在PDB液體培養(yǎng)基中所產毒素的種類和含量及洛伐他汀處理對其含量的影響;并結合蛇孢菌素處理后高粱葉片保護酶活性及質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,利用qRT-PCR技術對不同條件下該病原菌毒素合成關鍵酶編碼基因(IPPI/FPPS/HMGR/GGPPS)的表達進行了檢測。實驗結果如下: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胞中的侵染情況大致相同,均可從細胞表面和細胞間隙兩個途徑進入細胞內部。2.高粱靶斑病菌所產毒素以蛇孢菌素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胞中的侵染,導致病原菌的致病性顯著下降。且在0-1500μg/ml濃度范圍內,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)傷接種均可恢復病原菌的致病性。此外該毒素可引起高粱葉組織細胞膜透性改變,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間的延長細胞膜透性和MDA含量持續(xù)增大。同時還可造成高粱葉片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胞膜透性,造成膜功能的紊亂,同時影響寄主保護酶活,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關鍵酶。我們通過Real Time PCR技術,檢測了高粱靶斑病菌菌絲階段這四個酶的編碼基因(IPPI、FPPS、GGPPS、HGMR)在不同條件下的基因表達情況。結果顯示:不同濃度洛伐他汀處理(25μM、50μM、100μM、200μM)均可導致毒素合成酶編碼基因表達的下降,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)在不同處理時間(2h、8h、24h、36h、48h)條件下,隨著處理時間的推移,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的表達在任一時間均明顯下降。此外:我們發(fā)現(xiàn)不同溫度、光照、PH條件下IPPI、FPPS、GGPPS表達模式相似,適當?shù)母邷?32℃)均可提高這三種基因的表達;HMGR的表達模式則與其相反,高溫(32℃)反而不利于該基因的表達,PH4.0的弱酸環(huán)境則促進其表達,相比對照組升高近20倍,且HMGR基因對酸性環(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.
【學位授予單位】：河南師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S435.14

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