盾壳霉氮代谢调控基因cmareA的功能研究

发布时间：2018-10-18 08:34

【摘要】：核盘菌(Sclerotinia sclerotiorum(Lib.)de Bary)是油菜菌核病的病原菌,每年造成油菜大量减产,经济损失巨大。重寄生真菌盾壳霉(Coniothyrium minntans)对核盘菌专性寄生,对核盘菌的生物防治有着广阔的开发前景。实验室前期研究发现盾壳霉能降解核盘菌产生的草酸毒素,这一过程中可以通过产生氨来缓解酸化的环境,也说明这其中伴随着盾壳霉的氮源代谢调控,而参与该氮源代谢的调控基因和功能还未见报道。盾壳霉氮源代谢调控基因(cmareA)对其生长发育,盾壳霉重寄生,降解草酸和产生抗真菌的影响也尚不清楚。因此,该课题在实验室前期研究的基础上,对盾壳霉的氮源代谢调控基因cmareA进行了深入研究,获得的主要研究成果如下:从盾壳霉Chy-1中克隆获得cmareA部分序列,比对盾壳霉基因组数据库获得全部cmareA基因序列,基因全长2811 bp,除去一个内含子之后的外显子序列大小为2754 bp,该开放阅读框编码917个氨基酸。系统进化树分析结果表明cmareA编码的氨基酸与小麦颖枯病菌(Parastagonospora nodorum)的同源areA编码的氨基酸亲缘关系最近,同源性高达85%。基因的表达分析证明cmareA基因只有在优先氮源不足或氮源饥饿状态下,cmareA的转录调控才会被激活,cmareA基因才会表达。通过Split-Marker技术对cmareA基因进了敲除与互补,获得敲除突变体ΔcmareA-129和ΔcmareA-133,相对应的互补转化子ΔcmareA-129C和ΔcmareA-133C。对其生物学特性的研究发现,cmareA的缺失会减慢盾壳霉在PDA培养条件下的菌丝生长发育,同时会减低其产孢量。另外,cmareA的缺失会导致盾壳霉在以硝酸和铵盐为氮源的MCD培养条件下的菌丝生长受到抑制,生长速率显著下降。通过平板对峙试验和菌核沙皿寄生菌核试验,测定各突变体寄生核盘菌的能力。结果显示,cmareA敲除后,敲除转化子ΔcmareA-129和ΔcmareA-133对核盘菌菌丝和菌核的寄生能力减弱。进一步测定重寄生相关酶(胞外蛋白酶、葡聚糖酶和几丁质酶)活性,结果发现敲除突变体的重寄生能力的减弱可能是由于cmareA缺失导致几丁质酶活性降低所引起的。通过液相色谱的方法,测定各突变体降解草酸的能力,结果显示,cmareA缺失后盾壳霉降解草酸的能力也同样减弱。在不同氮源的培养条件,发现可能由于cmareA敲除突变体的生长速度减慢导致产生的抗真菌物质的能力也有不同程度的降低。另外,在研究氮源与pH对盾壳霉抗菌作用的交互影响实验中,发现盾壳霉以硝酸盐为氮源时产生AFS,酸性pH是必须的,cmareA也是必须的。综上所述,盾壳霉氮源代谢调控基因cmareA不仅参与了盾壳霉的生长发育、硝酸和铵类氮源的调控,cmareA基因还能正调控着盾壳霉的重寄生,草酸的降解,同时还调控抗真菌物质的产生。
[Abstract]:Sclerotinia sclerotiorum (Lib.) de Bary) is the pathogen of sclerotinia sclerotiorum. (Coniothyrium minntans), a highly parasitic fungus, is a specific parasitic fungus to Sclerotinia sclerotiorum, and has a broad prospect for biological control of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Previous laboratory studies have found that the bacteria can degrade oxalic acid toxin produced by Sclerotinia sclerotiorum, which can alleviate the acidified environment by producing ammonia, which also shows that the nitrogen source metabolism regulation is accompanied by chitosan. However, the regulatory genes and functions involved in the metabolism of this nitrogen source have not been reported. The effects of (cmareA) on its growth and development, the high parasitism, the degradation of oxalic acid and the production of antifungal were also unclear. Therefore, on the basis of the previous research in laboratory, the cmareA gene regulating nitrogen source metabolism was deeply studied. The main research results were as follows: the partial sequence of cmareA was cloned from Chy-1. The whole cmareA gene sequence was obtained by comparing the genomic database of Trichoderma auratus. The exon sequence of the gene with a total length of 2811 bp, excluding an intron was 2754 bp,. The open reading frame encodes 917 amino acids. Phylogenetic tree analysis showed that the amino acid encoded by cmareA was closely related to the amino acid encoded by homologous areA of (Parastagonospora nodorum) of wheat glume blight, and the homology was as high as 85g. Gene expression analysis showed that cmareA gene can only be activated by cmareA transcription regulation and cmareA gene expression only when the priority nitrogen source is insufficient or the nitrogen source is hungry. The complementary transformants 螖 cmareA-129 and 螖 cmareA-133, corresponding to the knockout mutants 螖 cmareA-129 and 螖 cmareA-133, were obtained by Split-Marker. The complementary transformants 螖 cmareA-129C and 螖 cmareA-133C. were obtained. The study of its biological characteristics showed that the absence of cmareA could slow down the growth and development of the hyphae and reduce the sporulation under the condition of PDA culture. In addition, the absence of cmareA resulted in the inhibition of mycelium growth and the decrease of growth rate in MCD cultured with nitric acid and ammonium salt as nitrogen source. The ability of each mutant to parasitize Sclerotinia sclerotiorum was determined by plate confrontation test and nuclear test of Sclerotinia sclerotiorum parasite. The results showed that after cmareA knockout, the parasitic ability of knockout transformants 螖 cmareA-129 and 螖 cmareA-133 on sclerotia and sclerotia decreased. The activity of hyperparasitic related enzymes (extracellular protease, glucanase and chitinase) was further determined. It was found that the decrease in the ability of heavy parasitism of knockout mutants may be due to the decrease of chitinase activity due to the absence of cmareA. The ability of the mutants to degrade oxalic acid was determined by liquid chromatography. The results showed that the ability of chitosan to degrade oxalic acid was also weakened after the absence of cmareA. Under different nitrogen sources, it was found that the ability of antifungal substances produced by cmareA knockout mutants was also decreased in varying degrees due to the slow growth rate of the mutant. In addition, in the experiment of the interaction between nitrogen source and pH, it was found that it is necessary to produce AFS, acid pH when the nitrate is used as nitrogen source, and cmareA is also necessary. In conclusion, the nitrogen source regulation gene cmareA not only participates in the growth and development of chitosan, but also in the regulation of nitric acid and ammonium nitrogen source. CmareA gene can also regulate the hyperparasitism and the degradation of oxalic acid. At the same time, it also regulates the production of antifungal substances.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S476

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