生防菌盾壳霉LaeA基因的克隆与功能研究
发布时间：2018-08-13 16:39
【摘要】：核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)是一种世界广泛分布危害严重的病原真菌,至少能造成400多种植物发病。生防菌盾壳霉(Coniothyrium minntans)是核盘菌的一种重寄生真菌,对核盘菌引起的作物菌核病有很好的生防潜力。盾壳霉可以通过抗生作用或重寄生作用来控制核盘菌,而盾壳霉的抗生作用或重寄生作用受环境p H值的调控。Lae A是一种全局性的次级代谢调控因子,可以调控真菌的次级代谢和发育,例如在Aspergillus、Fusarium、Penicillium和Trichoderma属等真菌中已有报道。但在盾壳霉中还没有Lae A基因及其功能研究的相关报道。那么Lae A在盾壳霉的次级代谢和发育中有何作用?本研究采用简并引物同源扩增技术从盾壳霉基因组中克隆得到Cmlae A基因,并通过PEG介导的原生质体转化技术对Cmlae A基因进行敲除和互补,最后对Cmlae A在盾壳霉重寄生和抗生作用中的功能进行了研究,取得如下结果:首先,从盾壳霉野生型菌株Chy-1中克隆到Lae A基因及其启动子序列,将其命名为Cmlae A(Gen Bank登录号:KP998106)。Cmlae A基因全长2017bp,包含6个外显子,5个内含子,1749bp的开放阅读框共编码583个氨基酸。对其氨基酸序列进行分析发现,Cmlae A氨基酸序列中含有Lae A蛋白的保守结构域(S-腺苷甲硫氨酸结合域)。系统进化分析结果表明:Cmlae A与Pyrenophora tritici-repentis的Lae A亲缘关系最近,氨基酸同源性高达80%。q RT-PCR试验结果表明Cmlae A在24 h黑暗条件下增强表达,Cmlae A在24 h光照条件下的表达量仅为24 h黑暗条件下的0.179倍。其次,通过同源重组技术获得了Cmlae A的敲除突变体ΔCmlae A-2和互补转化子ΔCmlae A-2C。不论在光照还是黑暗条件下,各突变体在PDA平板上菌丝生长速率与野生型无显著差异,但Cmlae A敲除突变体不能产生分生孢子器与分生孢子,而且其菌落的表面疏水性丧失。可见,Cmlae A显著影响盾壳霉分生孢子器、分生孢子的产生和菌落表面疏水性,而且这些生物学特性不受光照调控。然后,明确了Cmlae A对盾壳霉重寄生作用的影响。重寄生试验结果表明,Cmlae A敲除突变体对核盘菌菌丝和菌核的寄生能力显著低于野生型和互补转化子ΔCmlae A-2C。进一步检测重寄生相关酶的产生情况,Cmlae A敲除突变体产生的胞外蛋白酶、几丁质酶和葡聚糖酶显著低于野生型和互补转化子ΔCmlae A-2C。因此,Cmlae A基因的敲除导致盾壳霉产生重寄生相关酶(胞外蛋白酶、几丁质酶和葡聚糖酶)的能力下降,从而显著降低了盾壳霉对核盘菌的重寄生能力。此外,还研究了Cmlae A对盾壳霉抗生作用的影响。在PDB摇培条件下,敲除突变体ΔCmlae A-2产生的抗真菌物质显著高于野生型和互补转化子ΔCmlae A-2C,同时Cmlae A敲除突变体培养滤液的p H值显著低于野生型和互补转化子ΔCmlae A-2C。在MCD缓冲p H值条件下,只有在p H值为5.0的条件下,Cmlae A敲除突变体的培养滤液几乎没有抑菌活性,显著低于野生型和互补转化子;而在p H为3.0、4.0和6.0时,Cmlae A敲除突变体培养滤液的抑菌活性与野生型和互补转化子没有显著差异,而且各盾壳霉菌株在高p H值条件产生抗真菌物质能力下降。因此,Cmlae A敲除突变体在PDB摇培条件下抗生活性增强可能是由于其培养液p H值降低所造成的。综上所述,Cmlae A在盾壳霉与核盘菌互作中发挥重要作用,不仅影响盾壳霉的无性发育(分生孢子器、分生孢子)和疏水性,而且还影响盾壳霉的生防功能-重寄生作用和抗生作用。
[Abstract]:Sclerotinia sclerotiorum (Lib.) de Bary is a kind of pathogenic fungi which is widely distributed in the world and can cause more than 400 kinds of plant diseases. Coniothyrium minntans is a heavy parasitic fungus of Sclerotinia sclerotiorum. It has a good biocontrol potential for Sclerotinia sclerotiorum-induced crop sclerotinia. Lae A is a global secondary metabolic regulator that can regulate secondary metabolism and development of fungi, such as Aspergillus, Fusarium, Penicillium and Trichoderma. In this study, Cmlae A gene was cloned from the genome of C. Scutellaria by degenerate primer homology amplification, and Cmlae A gene was transformed into protoplast mediated by PEG. At last, the function of Cmlae A in the heavy parasitism and antibiotic action of Puccinella Scutellaria was studied. The following results were obtained: Firstly, Lae A gene and its promoter sequence were cloned from wild-type strain Chy-1 of Puccinella Scutellaria and named Cmlae A (Gen Bank login number: KP998106). Cmlae A gene was 2017 BP in length, including 6 exons, 5,5. A total of 583 amino acids were encoded in an open reading frame of 1749 bp. The amino acid sequence of Cmlae A contained a conserved domain of Lae A protein (S-adenosylmethionine binding domain). Phylogenetic analysis showed that Cmlae A was closely related to Lae A of Pyrenophora tritici-repentions. The homology of Cmlae A was as high as 80%. The results of Q RT-PCR showed that the expression of Cmlae A was enhanced under 24 h darkness, and the expression of Cmlae A was 0.179 times higher than that under 24 h darkness. Secondly, the knockout mutant Cmlae A-2 and complementary transformant Cmlae A-2C were obtained by homologous recombination. Under the condition of PDA, the mycelial growth rate of each mutant was not significantly different from that of the wild type, but Cmlae A knockout mutant could not produce conidia and conidia, and the surface hydrophobicity of the colony was lost. The results of heavy parasitism test showed that the parasitism of Cmlae A knockout mutant to Sclerotinia sclerotiorum hypha and sclerotia was significantly lower than that of wild type and complementary transformant Cmlae A-2C. Extracellular proteases, chitinases and glucanases produced by knockout mutants were significantly lower than those produced by wild-type and complementary transformants Cmlae A-2C. Therefore, knockout of Cmlae A gene resulted in a decrease in the production of heavy parasitic related enzymes (extracellular proteases, chitinases and glucanases) by C. scutellaria, thereby significantly reducing the re-hosting of S. sclerotiorum to S. sclerotiorum. In addition, the effects of Cmlae A on the antifungal activities of Puccinella Scutellaria were studied. Under PDB shake culture, the antifungal substances produced by knockout mutant_Cmlae A-2 were significantly higher than those produced by wild type and complementary transformant_Cmlae A-2C, and the P H value of the filtrate of Cmlae A knockout mutant was significantly lower than that of wild type and complementary transformant_Cmlae A-2C. Under the condition of CD buffer P H, Cmlae A knockout mutant had almost no antimicrobial activity except at P H 5.0, which was significantly lower than that of wild-type and complementary transformants. However, at P H 3.0, 4.0 and 6.0, the antimicrobial activity of Cmlae A knockout mutant was not significantly different from that of wild-type and complementary transformants, and the antimicrobial activity of Cmlae A knockout mutant was not significantly different from that of wild-type and complementary transformants. In conclusion, Cmlae A plays an important role in the interaction between Sclerotinia sclerotiorum and Puccinella sclerotiorum, and not only affects the asexual development (meristem) of Puccinella sclerotiorum. Spore organelles, conidia) and hydrophobicity, but also affect the biocontrol function of Puccinella Scutellaria - heavy parasitism and antibiosis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12
本文编号：2181569
[Abstract]:Sclerotinia sclerotiorum (Lib.) de Bary is a kind of pathogenic fungi which is widely distributed in the world and can cause more than 400 kinds of plant diseases. Coniothyrium minntans is a heavy parasitic fungus of Sclerotinia sclerotiorum. It has a good biocontrol potential for Sclerotinia sclerotiorum-induced crop sclerotinia. Lae A is a global secondary metabolic regulator that can regulate secondary metabolism and development of fungi, such as Aspergillus, Fusarium, Penicillium and Trichoderma. In this study, Cmlae A gene was cloned from the genome of C. Scutellaria by degenerate primer homology amplification, and Cmlae A gene was transformed into protoplast mediated by PEG. At last, the function of Cmlae A in the heavy parasitism and antibiotic action of Puccinella Scutellaria was studied. The following results were obtained: Firstly, Lae A gene and its promoter sequence were cloned from wild-type strain Chy-1 of Puccinella Scutellaria and named Cmlae A (Gen Bank login number: KP998106). Cmlae A gene was 2017 BP in length, including 6 exons, 5,5. A total of 583 amino acids were encoded in an open reading frame of 1749 bp. The amino acid sequence of Cmlae A contained a conserved domain of Lae A protein (S-adenosylmethionine binding domain). Phylogenetic analysis showed that Cmlae A was closely related to Lae A of Pyrenophora tritici-repentions. The homology of Cmlae A was as high as 80%. The results of Q RT-PCR showed that the expression of Cmlae A was enhanced under 24 h darkness, and the expression of Cmlae A was 0.179 times higher than that under 24 h darkness. Secondly, the knockout mutant Cmlae A-2 and complementary transformant Cmlae A-2C were obtained by homologous recombination. Under the condition of PDA, the mycelial growth rate of each mutant was not significantly different from that of the wild type, but Cmlae A knockout mutant could not produce conidia and conidia, and the surface hydrophobicity of the colony was lost. The results of heavy parasitism test showed that the parasitism of Cmlae A knockout mutant to Sclerotinia sclerotiorum hypha and sclerotia was significantly lower than that of wild type and complementary transformant Cmlae A-2C. Extracellular proteases, chitinases and glucanases produced by knockout mutants were significantly lower than those produced by wild-type and complementary transformants Cmlae A-2C. Therefore, knockout of Cmlae A gene resulted in a decrease in the production of heavy parasitic related enzymes (extracellular proteases, chitinases and glucanases) by C. scutellaria, thereby significantly reducing the re-hosting of S. sclerotiorum to S. sclerotiorum. In addition, the effects of Cmlae A on the antifungal activities of Puccinella Scutellaria were studied. Under PDB shake culture, the antifungal substances produced by knockout mutant_Cmlae A-2 were significantly higher than those produced by wild type and complementary transformant_Cmlae A-2C, and the P H value of the filtrate of Cmlae A knockout mutant was significantly lower than that of wild type and complementary transformant_Cmlae A-2C. Under the condition of CD buffer P H, Cmlae A knockout mutant had almost no antimicrobial activity except at P H 5.0, which was significantly lower than that of wild-type and complementary transformants. However, at P H 3.0, 4.0 and 6.0, the antimicrobial activity of Cmlae A knockout mutant was not significantly different from that of wild-type and complementary transformants, and the antimicrobial activity of Cmlae A knockout mutant was not significantly different from that of wild-type and complementary transformants. In conclusion, Cmlae A plays an important role in the interaction between Sclerotinia sclerotiorum and Puccinella sclerotiorum, and not only affects the asexual development (meristem) of Puccinella sclerotiorum. Spore organelles, conidia) and hydrophobicity, but also affect the biocontrol function of Puccinella Scutellaria - heavy parasitism and antibiosis.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S476.12
【参考文献】
相关期刊论文 前3条
1 高同春，王振荣，胡宏云;油菜菌核病病原生物学特性研究[J];安徽农业科学;1995年04期
2 姜道宏，李国庆，易先宏，王道本;菌核寄生菌盾壳霉的研究 Ⅱ．不同菌株培养特性及寄生致腐菌核能力的比较[J];华中农业大学学报;1996年03期
3 iJ新美;油菜菌核病(Sclerotinia sclerotiorum)在我国的寄主范围及生态特性的娍查研究[J];植物病理学报;1959年02期
相关硕士学位论文 前1条
1 董慧;一株拮抗性芽孢杆菌的鉴定及防治莴苣菌核病效果和机制研究[D];华中农业大学;2008年
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