生防菌盾殼霉LaeA基因的克隆與功能研究
發(fā)布時間：2018-08-13 16:39
【摘要】：核盤菌(Sclerotinia sclerotiorum(Lib.)de Bary)是一種世界廣泛分布危害嚴(yán)重的病原真菌,至少能造成400多種植物發(fā)病。生防菌盾殼霉(Coniothyrium minntans)是核盤菌的一種重寄生真菌,對核盤菌引起的作物菌核病有很好的生防潛力。盾殼霉可以通過抗生作用或重寄生作用來控制核盤菌,而盾殼霉的抗生作用或重寄生作用受環(huán)境p H值的調(diào)控。Lae A是一種全局性的次級代謝調(diào)控因子,可以調(diào)控真菌的次級代謝和發(fā)育,例如在Aspergillus、Fusarium、Penicillium和Trichoderma屬等真菌中已有報道。但在盾殼霉中還沒有Lae A基因及其功能研究的相關(guān)報道。那么Lae A在盾殼霉的次級代謝和發(fā)育中有何作用?本研究采用簡并引物同源擴增技術(shù)從盾殼霉基因組中克隆得到Cmlae A基因,并通過PEG介導(dǎo)的原生質(zhì)體轉(zhuǎn)化技術(shù)對Cmlae A基因進(jìn)行敲除和互補,最后對Cmlae A在盾殼霉重寄生和抗生作用中的功能進(jìn)行了研究,取得如下結(jié)果:首先,從盾殼霉野生型菌株Chy-1中克隆到Lae A基因及其啟動子序列,將其命名為Cmlae A(Gen Bank登錄號:KP998106)。Cmlae A基因全長2017bp,包含6個外顯子,5個內(nèi)含子,1749bp的開放閱讀框共編碼583個氨基酸。對其氨基酸序列進(jìn)行分析發(fā)現(xiàn),Cmlae A氨基酸序列中含有Lae A蛋白的保守結(jié)構(gòu)域(S-腺苷甲硫氨酸結(jié)合域)。系統(tǒng)進(jìn)化分析結(jié)果表明:Cmlae A與Pyrenophora tritici-repentis的Lae A親緣關(guān)系最近,氨基酸同源性高達(dá)80%。q RT-PCR試驗結(jié)果表明Cmlae A在24 h黑暗條件下增強表達(dá),Cmlae A在24 h光照條件下的表達(dá)量僅為24 h黑暗條件下的0.179倍。其次,通過同源重組技術(shù)獲得了Cmlae A的敲除突變體ΔCmlae A-2和互補轉(zhuǎn)化子ΔCmlae A-2C。不論在光照還是黑暗條件下,各突變體在PDA平板上菌絲生長速率與野生型無顯著差異,但Cmlae A敲除突變體不能產(chǎn)生分生孢子器與分生孢子,而且其菌落的表面疏水性喪失�？梢�,Cmlae A顯著影響盾殼霉分生孢子器、分生孢子的產(chǎn)生和菌落表面疏水性,而且這些生物學(xué)特性不受光照調(diào)控。然后,明確了Cmlae A對盾殼霉重寄生作用的影響。重寄生試驗結(jié)果表明,Cmlae A敲除突變體對核盤菌菌絲和菌核的寄生能力顯著低于野生型和互補轉(zhuǎn)化子ΔCmlae A-2C。進(jìn)一步檢測重寄生相關(guān)酶的產(chǎn)生情況,Cmlae A敲除突變體產(chǎn)生的胞外蛋白酶、幾丁質(zhì)酶和葡聚糖酶顯著低于野生型和互補轉(zhuǎn)化子ΔCmlae A-2C。因此,Cmlae A基因的敲除導(dǎo)致盾殼霉產(chǎn)生重寄生相關(guān)酶(胞外蛋白酶、幾丁質(zhì)酶和葡聚糖酶)的能力下降,從而顯著降低了盾殼霉對核盤菌的重寄生能力。此外,還研究了Cmlae A對盾殼霉抗生作用的影響。在PDB搖培條件下,敲除突變體ΔCmlae A-2產(chǎn)生的抗真菌物質(zhì)顯著高于野生型和互補轉(zhuǎn)化子ΔCmlae A-2C,同時Cmlae A敲除突變體培養(yǎng)濾液的p H值顯著低于野生型和互補轉(zhuǎn)化子ΔCmlae A-2C。在MCD緩沖p H值條件下,只有在p H值為5.0的條件下,Cmlae A敲除突變體的培養(yǎng)濾液幾乎沒有抑菌活性,顯著低于野生型和互補轉(zhuǎn)化子;而在p H為3.0、4.0和6.0時,Cmlae A敲除突變體培養(yǎng)濾液的抑菌活性與野生型和互補轉(zhuǎn)化子沒有顯著差異,而且各盾殼霉菌株在高p H值條件產(chǎn)生抗真菌物質(zhì)能力下降。因此,Cmlae A敲除突變體在PDB搖培條件下抗生活性增強可能是由于其培養(yǎng)液p H值降低所造成的。綜上所述,Cmlae A在盾殼霉與核盤菌互作中發(fā)揮重要作用,不僅影響盾殼霉的無性發(fā)育(分生孢子器、分生孢子)和疏水性,而且還影響盾殼霉的生防功能-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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.12
本文編號：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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S476.12
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