本文選題：香蕉枯萎病菌 + 枯草芽孢桿菌　； 參考：《華南農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：香蕉枯萎病是由尖孢鐮刀菌古巴專化型(Fusarium oxysporum f.sp.cubense)引起的維管束系統(tǒng)性病害,對香蕉生產(chǎn)造成毀滅性的破壞,引起了巨大的經(jīng)濟(jì)損失。香蕉枯萎病菌4號小種(Foc4)幾乎能感染所有的香蕉品種,為害嚴(yán)重。目前,尚無有效的方法防治香蕉枯萎病。利用拮抗細(xì)菌進(jìn)行生物防治的研究,近年來引起人們的關(guān)注。本研究室前期分離的枯草芽孢桿菌菌株G-7(Bacillus subtilis subsp.subtilis G-7)能有效抑制Foc4的菌絲生長和孢子萌發(fā),具有較高的生防潛力。本研究試圖從拮抗基因的克隆和表達(dá)方面,為香蕉枯萎病的防治提供理論參考和生防材料�？莶菅挎邨U菌與病原真菌作用時(shí),會(huì)分泌脂肽類抗生素(Lipopeptide antibiotic),其中依枯草菌素A(Iturin A)能干擾病原真菌細(xì)胞質(zhì)膜功能導(dǎo)致細(xì)胞死亡。本研究分析了枯草芽孢桿菌G-7對Foc4抑菌活性,對iturin A合成相關(guān)基因lpa-14、itu D-1進(jìn)行了克隆和序列分析,并研究lpa-14、itu D-1在菌株G-7和Foc4拮抗后的表達(dá)情況。在菌株G-7與Foc4平板對峙培養(yǎng)中,菌株G-7對Foc4的抑菌帶寬度達(dá)到5.90±0.10(mm),抑制率達(dá)到61.24±0.39(%)。溫室防效試驗(yàn)結(jié)果表明:巴西蕉(Musa AAA)蕉苗經(jīng)浸根接種菌株G-7培養(yǎng)液后,再植回含有Foc4孢子液土壤的盆中,30 d后觀察結(jié)果,陽性對照蕉苗表現(xiàn)出典型的香蕉枯萎病癥狀,而經(jīng)過菌株G-7培養(yǎng)液處理的巴西蕉苗發(fā)病情況明顯低于對照,顯示了良好的苗期活體抑菌效果。說明G-7菌株對Foc4有明顯的拮抗效果。通過生長速率法對菌株G-7的發(fā)酵液、菌體懸浮液和培養(yǎng)濾液處理下Foc4菌絲的抑制率研究發(fā)現(xiàn),菌株G-7能夠抑制Foc4的菌絲體生長。菌株G-7的發(fā)酵液、菌體懸浮液和培養(yǎng)濾液對Foc4菌絲體的抑制率分別為69.15±0.49(%),59.54±0.13(%)和31.83±0.08(%),均顯著高于對照組(P0.05)。通過孢子萌發(fā)法,對菌株G-7發(fā)酵液、菌體懸浮液和培養(yǎng)濾液處理下Foc4孢子萌發(fā)研究發(fā)現(xiàn),3種處理液對Foc4孢子的萌發(fā)具有明顯的抑制作用,菌株G-7發(fā)酵液、菌體懸浮液和培養(yǎng)濾液處理下的Foc4孢子萌發(fā)率分別為23.28±0.10(%),44.42±3.82(%)和32.01±0.17(%),顯著低于對照組(P0.05)�？寺×司闓-7 2個(gè)與脂肽類抗生素iturin A合成相關(guān)的基因itu D-1和lpa-14。測序得出,itu D-1和lpa-14的DNA分別為1202 bp和675 bp。通過NCBI相似性分析發(fā)現(xiàn),lpa-14編碼蛋白與Bacillus amyloliquefaciens(WP_060657811.1)中的磷酸泛酰巰基乙胺基轉(zhuǎn)移酶(PPTase)相似性為99%,且蛋白功能域分析發(fā)現(xiàn)lpa-14編碼蛋白屬于ACPS家族中的Sfp型蛋白;itu D-1編碼蛋白與Bacillus amyloliquefaciens(CBI43040.1)的丙二酰單酰輔酶A轉(zhuǎn)酰基酶(MCAT)相似性為99%,屬于Acy1_transf_1家族,是Fab D型蛋白中的抗生素生物合成蛋白質(zhì)。熒光定量PCR表明Foc4與菌株G-7拮抗24 h后的itu D-1和lpa-14的表達(dá)量較未處理組明顯上調(diào),其中l(wèi)pa-14較未處理組表達(dá)量上調(diào)了2.3倍,itu D-1上調(diào)了14.62倍。說明lpa-14、itu D-1在菌株G-7對Foc4拮抗過程中可能有重要的作用,是拮抗相關(guān)基因。
[Abstract]:Banana wilt disease by Fusarium oxysporum f.sp. (Fusarium oxysporum f.sp.cubense) of vascular system disease caused by the devastating damage to banana production, causing huge economic losses. FOC race 4 (Foc4) infection can almost all banana varieties, serious damage. At present, there is no the effective methods for prevention and treatment of banana wilt disease. Research on biological control by antagonistic bacteria, causing the attention of people in recent years. Our lab isolated Bacillus subtilis strain G-7 (Bacillus subtilis subsp.subtilis G-7) can effectively inhibit the mycelial growth and spore germination of Foc4, has high biocontrol potential. This study attempts to clone antagonistic gene and expression, and provide a theoretical reference for the prevention and control of materials and biocontrol of Fusarium wilt disease of banana. Bacillus subtilis and pathogenic fungi effect, will be divided into resinosis peptide (Lipopeptide antibiotic), the antibiotic iturin A (Iturin A) can interfere with cell membrane function of pathogenic fungi causing cell death. This study analysis of Bacillus subtilis G-7 on the inhibitory activity of Foc4 on iturin, A lpa-14 ITU D-1 synthesis related genes, were cloned and sequenced, and lpa-14, ITU in D-1 the expression of G-7 and Foc4 strains after antagonistic strain G-7 and Foc4. In the dual culture, strain G-7 of Foc4 inhibitory band width to 5.90 + 0.10 (mm), the inhibition rate was 61.24 + 0.39 (%). The greenhouse control effect test results show that: Brazil banana (Musa AAA) by dipping the root of Banana Seedlings the inoculated strains cultured in G-7 after reimplantation with Foc4 spores in soil in the basin, after 30 d observation results, the positive control of banana seedlings showed typical symptoms of Fusarium Wilt of banana, and after strain G-7 culture fluid of the Brazil banana seedlings incidence was significantly lower than the control, significant The seedling in good antibacterial effect. The results showed that G-7 strain had antagonistic effect on Foc4. The growth rate of strain G-7 fermentation broth, cell suspension and culture filtrate of Foc4 inhibited mycelium growth rate of mycelium, strain G-7 could inhibit Foc4. The fermentation liquid of strain G-7, strain suspension culture filtrate of Foc4 and inhibit the mycelium rate were 69.15 + 0.49 (%), 59.54 + 0.13 and 31.83 + 0.08 (%) (%), were significantly higher than control group (P0.05). The spore germination method of strain G-7 fermentation, cell suspension and culture filtrate treatment under Foc4 the study found that the spore germination, 3 kinds of solution on the germination of Foc4 spores have obvious inhibition effect, strain G-7 fermentation, cell suspension culture and Foc4 spore germination under leachate treatment were 23.28 + 0.10 (%), 44.42 + 3.82 and 32.01 + 0.17 (%) (%) was significantly lower than the control. Group (P0.05). The cloned strain G-7 2 and that of lipopeptide antibiotic iturin A synthesis related genes ITU D-1 and lpa-14. ITU D-1 sequencing, and lpa-14 DNA were 1202 BP and 675 bp. by NCBI similarity analysis indicated that lpa-14 encoding protein with Bacillus amyloliquefaciens (WP_060657811.1) enzyme phosphopantetheinyl in the transfer of (PPTase) similarity was 99%, and the protein functional domain analysis showed that lpa-14 encoding protein belongs to Sfp type proteins in the ACPS family; ITU D-1 encoding protein with Bacillus amyloliquefaciens (CBI43040.1) two propylene acyl CoA A acyltransferase (MCAT) similarity is 99%, belonging to the Acy1_transf_1 family, is a protein Fab type D protein in antibiotic biosynthesis. Fluorescence quantitative PCR showed that the expression levels of Foc4 and G-7 strains antagonistic after 24 h ITU D-1 and lpa-14 was significantly increased compared with untreated group, the expression of lpa-14 compared with the untreated group The amount raised 2.3 times, ITU D-1 raised 14.62 times. Lpa-14, ITU D-1 in strain G-7 of Foc4 antagonistic process may have an important role, is the antagonistic related genes.

【學(xué)位授予單位】：華南農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S436.68

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相關(guān)期刊論文 前10條

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本文編號：1752352