枯草芽孢杆菌G-7拮抗香蕉枯萎病菌的效果及相关基因研究

发布时间：2018-04-15 03:32

本文选题：香蕉枯萎病菌 + 枯草芽孢杆菌　；参考：《华南农业大学》2016年硕士论文

【摘要】：香蕉枯萎病是由尖孢镰刀菌古巴专化型(Fusarium oxysporum f.sp.cubense)引起的维管束系统性病害,对香蕉生产造成毁灭性的破坏,引起了巨大的经济损失。香蕉枯萎病菌4号小种(Foc4)几乎能感染所有的香蕉品种,为害严重。目前,尚无有效的方法防治香蕉枯萎病。利用拮抗细菌进行生物防治的研究,近年来引起人们的关注。本研究室前期分离的枯草芽孢杆菌菌株G-7(Bacillus subtilis subsp.subtilis G-7)能有效抑制Foc4的菌丝生长和孢子萌发,具有较高的生防潜力。本研究试图从拮抗基因的克隆和表达方面,为香蕉枯萎病的防治提供理论参考和生防材料。枯草芽孢杆菌与病原真菌作用时,会分泌脂肽类抗生素(Lipopeptide antibiotic),其中依枯草菌素A(Iturin A)能干扰病原真菌细胞质膜功能导致细胞死亡。本研究分析了枯草芽孢杆菌G-7对Foc4抑菌活性,对iturin A合成相关基因lpa-14、itu D-1进行了克隆和序列分析,并研究lpa-14、itu D-1在菌株G-7和Foc4拮抗后的表达情况。在菌株G-7与Foc4平板对峙培养中,菌株G-7对Foc4的抑菌带宽度达到5.90±0.10(mm),抑制率达到61.24±0.39(%)。温室防效试验结果表明:巴西蕉(Musa AAA)蕉苗经浸根接种菌株G-7培养液后,再植回含有Foc4孢子液土壤的盆中,30 d后观察结果,阳性对照蕉苗表现出典型的香蕉枯萎病症状,而经过菌株G-7培养液处理的巴西蕉苗发病情况明显低于对照,显示了良好的苗期活体抑菌效果。说明G-7菌株对Foc4有明显的拮抗效果。通过生长速率法对菌株G-7的发酵液、菌体悬浮液和培养滤液处理下Foc4菌丝的抑制率研究发现,菌株G-7能够抑制Foc4的菌丝体生长。菌株G-7的发酵液、菌体悬浮液和培养滤液对Foc4菌丝体的抑制率分别为69.15±0.49(%),59.54±0.13(%)和31.83±0.08(%),均显著高于对照组(P0.05)。通过孢子萌发法,对菌株G-7发酵液、菌体悬浮液和培养滤液处理下Foc4孢子萌发研究发现,3种处理液对Foc4孢子的萌发具有明显的抑制作用,菌株G-7发酵液、菌体悬浮液和培养滤液处理下的Foc4孢子萌发率分别为23.28±0.10(%),44.42±3.82(%)和32.01±0.17(%),显著低于对照组(P0.05)。克隆了菌株G-7 2个与脂肽类抗生素iturin A合成相关的基因itu D-1和lpa-14。测序得出,itu D-1和lpa-14的DNA分别为1202 bp和675 bp。通过NCBI相似性分析发现,lpa-14编码蛋白与Bacillus amyloliquefaciens(WP_060657811.1)中的磷酸泛酰巯基乙胺基转移酶(PPTase)相似性为99%,且蛋白功能域分析发现lpa-14编码蛋白属于ACPS家族中的Sfp型蛋白;itu D-1编码蛋白与Bacillus amyloliquefaciens(CBI43040.1)的丙二酰单酰辅酶A转酰基酶(MCAT)相似性为99%,属于Acy1_transf_1家族,是Fab D型蛋白中的抗生素生物合成蛋白质。荧光定量PCR表明Foc4与菌株G-7拮抗24 h后的itu D-1和lpa-14的表达量较未处理组明显上调,其中lpa-14较未处理组表达量上调了2.3倍,itu D-1上调了14.62倍。说明lpa-14、itu D-1在菌株G-7对Foc4拮抗过程中可能有重要的作用,是拮抗相关基因。
[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.

【学位授予单位】：华南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S436.68

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