【作者】 韩永超；

【导师】 李国庆；

【作者基本信息】 华中农业大学 ， 植物病理学， 2010， 博士

【摘要】 盾壳霉(Coniothyrium minitans)是核盘菌(Sclerotinia sclerotiorum)的重寄生真菌,在世界范围内广泛分布,在菌核病的生物防治中有着非常重要的作用。本论文主要针对碳源,氮源和环境pH对盾壳霉产生抗真菌物质(AFS)的调控及其分子机理进行了研究。研究发现盾壳霉菌株Chy-1在改良的查比培养基(MCD)中培养时能稳定产生AFS；而在马铃薯葡萄糖液体培养基(PDB)中培养时却不能产生AFS。为了进一步研究盾壳霉在MCD和PDB培养液中生长时产生AFS的差异,研究了营养和环境pH值对盾壳霉产生AFS的影响。结果显示：盾壳霉培养物滤液对核盘菌菌丝生长的抑制率与环境pH值相关,较适宜盾壳霉产生AFS的碳源或氮源,其培养液的环境pH值都较低。当盾壳霉在环境pH值缓冲为3的培养液中生长时,培养滤液对核盘菌菌丝生长的抑制率远远高于当环境pH值缓冲为6的培养液。为了研究环境pH值调控盾壳霉产生抗真菌物质的分子机理,本文用简并引物和RACE技术从盾壳霉菌株Chy-1中克隆到了pH感应转录调控因子基因(cmpacC)和碳代谢抑制因子基因(cmcreA)。cmpacC的全长序列(GenBank Ace. No. FJ176399)含有一个开放阅读框和两个内含子。根据cmpacC的碱基序列推测cmpacC编码一个长度为596个氨基酸残基的蛋白质(GenBank Ace. No. ACN43303),其含有三个保守的锌指结构域。同时本文也用基因组步移的方法克隆到了长度为2740bp的cmpacC 5’端侧翼序列及长度为2730bp的cmpacC 3’端侧翼序列。其中,5’端侧翼序列含有5个PacC结合位点(5’-GCCAAG-3’)和三个CreA结合位点(5’-SYGGRG-3’)。确定了cmpacC转录时的加A位点。荧光定量RT-PCR的分析结果显示：当盾壳霉在不同环境pH条件下生长时,cmpacC、几丁质酶基因(cmchl)(?)口p-1,3-葡聚糖酶基因(cmgl)的表达量都随着环境pH值的上升而升高。当盾壳霉在以单糖为碳源的培养基中生长时,cmcreA基因的表达量低,而当碳源为微生物不易利用的多糖时,cmcreA的相对表达量高。利用农杆菌介导同源重组的方法对盾壳霉中cmpalF基因进行了敲除。结果显示：当cmpalF基因缺失后,cmpacC的表达量降低,在环境pH高于6的环境中生长速度显著低于野生型菌株。在缓冲环境pH值为6时,培养滤液对核盘菌菌丝生长的抑制率显著高于野生型菌株。用数字化表达谱技术分析了盾壳霉菌株Chy-1在环境pH值为3和6条件下RNA转录水平的表达差异基因。共得到了1286个差异表达的基因,以盾壳霉RNA样品Cm3的基因表达量为参照时,RNA样品Cm6中有596个基因上调表达,690个基因下调表达。上述结果为解释盾壳霉与核盘菌之间的互作以及进一步揭示盾壳霉产生AFS的分子机制奠定了基础。更多还原

【Abstract】 Coniothyrium minitans (CM) is a promising biocontrol agent of Sclerotinia spp. including Sclerotinia sclerotiorum (SS). My research work mainly focused on carbon, nitrogen and ambient pH regulate the production of antifungal substances (AFS) by C. minitans and the molecular mechanism.We found that the strain Chy-1 of C. minitans used in our lab could consistently produce AFS in modified Czapek-Dox (MCD) broth; however, production of AFS by this strain of C. minitans was hardly detected in potato dextrose broth (PDB). In order to elucidate the difference of antifungal activity (AA) of the mycoparasite C. minitans against S. sclerotiorum (AAcm-ss) of MCDcm and PDBcm, we studied the effects of ambient pH and nutritional factors on antifungal activity of C. minitans. The results showed that production of AACM-ss by C. minitans was closely related to ambient pH. The carbon or nitrogen suit for AFS production was closely related to low ambient pH of the medium. AACM-ss was much higher in C. minitans cultures buffered at pH 3 than that buffered at pH 6.For the molecular mechanism of ambient pH regulate AFS production in C. minitans. We cloned a putative pH-responsive transcription factor gene (cmpacC) and a putative carbon repression gene (cmcreA) from strain Chy-1 of C. minitans by degenerate PCR and Rapid amplification of cDNA ends. The full-length DNA sequence of cmpacC (GenBank Ace. No. FJ176399) has one open reading frame (ORF) and two introns. cmpacC putatively encodes a polypeptide with 596 amino acid residues (GenBank Ace. No. ACN43303). The sequence of this polypeptide has three conserved zinc finger domains. Meanwhile, a 2,740 bp-long 5’ flanking sequence of cmpacC and a 2,730bp-long 3’flanking sequence of cmpacC was obtained by genome walking. It contains five copies of PacC binding sites (5’-GCCAAG-3’). Real-time RT-PCR analysis indicated that with the increase of ambient pH from 3 to 8, expression of cmpacC, as well as the chitinase gene (cmchl) and theβ-1,3-glucanase gene (cmgl), increased consistently in C. minitans. When C.minitans grows in the media with the carbon source of Monosaccharide, the expression of cmcreA is relative lower than grow in the media with the carbon source of polysaccharide. We knocked out the cmpalF gene in C. minitans by Agrobacterium tumefacies-mediated homologous recombination. We found that when ambient pH of the media was higher than 6, cmpalF grew more slowly than the wild type strain Chy-1. AACM-ss of AcmpalF mutant was higher than Chy-1. The expression of cmpacC in cmpalF was significantly lower than that in strain Chy-1. We analysed the differentially expressed genes of C. minitans grew in the media of different ambient pH (pH 3 and pH 6). There were a total of 1286 genes of C. minitans differentially expressed,596 genes up-regulated and 690 genes down-regulated.These results broadened our understanding of the interaction between C. minitans and S. sclerotiorum and laid a solid foundation for future studies to elucidate molecular mechanisms involved in AFS biosynthesis in C. minitans.更多还原