少孢节丛孢中7个生物合成基因对真菌表型、捕食及次级代谢影响的初步探究

发布时间：2018-01-20 08:54

本文关键词： 少孢节丛孢生物合成基因次级代谢产物表型捕食线虫能力　出处：《云南大学》2016年硕士论文　论文类型：学位论文

【摘要】：少孢节丛孢(Arthrobotrys oligospora)能产生丰富的次级代谢产物,但目前对其次级代谢产物生物合成基因的研究却甚少。本论文根据少孢节丛孢中全基因组的生物合成基因的分析预测结果,筛选出7个生物合成基因作为研究对象,其中包括Ⅰ型聚酮合酶(PKS)基因AOL_s00043g828,Ⅲ型PKS基因AOL_s00043g287,吲哚生物合成基因簇上的异戊烯基转移酶基因AOL_s00043g400, P450基因AOLs00043g381、AOL_s000789577.AOL_s00097g233.AOL_s00097g384。利用遗传转化手段,获得了这7个基因的突变菌株,并对7个基因的突变株和野生菌株在菌落形态、菌落直径生长、产孢量、捕食线虫能力及次级代谢产物等方面进行了比较,此外,还比较了其中2个基因的突变株和野生菌株的基因表达差异。表型比较发现,P450基因AOL_s00043g381和AOL_s00078g577突变株与野生菌株相比产生了较明显的变化。两株突变菌株菌落生长均比野生菌株快,其中ΔOL_s00043g381突变株在PDA培养基中1-6 d增长幅度达到了24%-88%不等,ΔAOL_s00078g577突变株在YMA培养基中1-5 d提高了11-22%；但ΔAOL_s00043g381突变株气生菌丝比野生菌株少,而ΔAOL_s00078g577突变株与野生菌株相比菌丝生长明显茂盛；在产孢量上,ΔAOL__s00043g381突变株比野生菌株减少了约2.5倍,而ΔAOL_s00078g577突变株产孢量增加了5.6倍：在捕器数量上,ΔAOL_s00078g577在12,18,24和36 h,与野生菌株相比分别高出13,14,2.3和3倍：在杀线虫能力上,ΔAOL_s00078g577突变株与野生菌株相比在12,18和24 h,分别高出60,9.6和2.1倍。其他基因突变株与野生菌株相比,在形态,捕食线虫能力上无显著差异。代谢产物分析发现,与野生菌株发酵液粗提物的HPLC图谱相比,Ⅰ型PKS基因AOL_s00043g828突变株和吲哚生物合成基因簇上的异戊烯基转移酶基因AOL_s00043g400突变株,均有6个峰的面积增大,其中面积明显增高的峰在ΔAOL_s00043g828突变株中有4个,ΔAOL_s00043g400突变株中有3个, 此外,在ΔAOL_s00043g400突变株中还有5个峰面积减小,4个峰消失。GC-MS检测结果显示,ΔAOL_s00043g828突变菌株中的化合物要比野生菌株丰富,突变株中特有的化合物有7个,含量明显增高的峰有3个,而野生菌株中只有3个特有化合物。在ΔAOL_s00043g400突变株中,发现在野生菌株中特有7个化合物中有5个为含有异戊烯基单位的化合物,在变株中仅有1个含异戊烯基单位的化合物和1个酰胺类化合物,在突变株中发现含量增加化合物中有1个氨基酸类化合物。同时发现P450基因ΔAOL_s00043g381、AOL_s00097g384突变株与野生菌株相比,发生变化的主要是长链脂肪烷烃和长链脂肪酯类化合物。此外,Ⅲ型PKS基因AOL_s00043g287突变株,P450基因AOL_s00078g577和AOL_s00097g233突变株发酵液粗提物的HPLC及GC-MS图谱与野生菌株相比均无差异。基因表达谱分析发现Ⅰ型PKS基因AOL_s00043g828突变株有151个显著差异表达基因,表达下调基因94个,表达上调基因57个。在P450基因突变菌株中,AOL_s00097g384突变株中引起的差异表达基因远远多于ΔAOL_s00043g828突变株,共有1231个显著差异表达基因,表达下调基因有743个,表达上调基因488个。根据上述实验结果发现,在本论文研究的7个生物合成基因中,对代谢产物有影响的基因类型有Ⅰ型PKS基因、异戊烯基转移酶基因和P450基因,对表型和捕食线虫能力产生影响的都是P450基因。在4个P450基因中,AOL_s00043g381和AOL_s00078g577能影响菌落生长及产孢,尤其是AOL_s00078g577对捕器形成和线虫捕食率有显著影响,此外,ΔAOL_s00043g381和AOL_s00097g384对代谢产物产生了一定影响,且AOL_s00097g384基因的敲除引起了1231个基因表达发生了显著性变化。以上结果表明P450基因对少孢节丛孢菌株生长发育及代谢有重要作用,且首次发现P450基因能参与捕食线虫真菌捕器形成及捕食线虫能力的调控。此外,根据研究结果推测,在少孢节丛孢中,异戊烯基转移酶基因AOL_s00043g400可能参与吲哚类化合物生物合成的异戊烯基转移过程,Ⅲ型PKS基因AOL_s00043g287及P450基因AOL_s00043g287在本实验条件下可能处于沉默状态。总之,在少孢节丛孢中,部分生物合成基因能对真菌表型、捕食能力及次级代谢产物产生影响,但是这些生物合成基因是如何发挥作用,次级代谢产物与表型及捕食线虫之间存在何种联系还不清楚,有待进一步探究。本论文为研究次级代谢产物多样性及基因多样性提供了菌株,为进一步开展少孢节丛孢中生物合成基因的研究奠定了基础。
[Abstract]:A.oligosporus (Arthrobotrys oligospora) can produce abundant secondary metabolites, but there is little research on its secondary metabolites biosynthesis gene. The prediction results based on the analysis of Arthrobotrys oligospora Baozhong genome biosynthetic genes, screened 7 biosynthetic genes as the research object, including the type of poly ketone synthase (PKS) gene AOL_s00043g828, PKS gene of AOL_s00043g287 type III, prenylated indole biosynthesis gene cluster transferase gene AOL_s00043g400, P450 gene AOLs00043g381, AOL_s000789577.AOL_s00097g233.AOL_s00097g384. by genetic transformation method, the mutant strains of these 7 genes, and 7 genes in the mutant and wild strains in colony morphology colony diameter, growth, sporulation, nematode trapping ability and secondary metabolites were compared, in addition, also compared The expression of 2 genes in the mutant and wild strains of the genetic differences. Phenotypic comparison, P450 gene AOL_s00043g381 and AOL_s00078g577 mutant compared with the wild isolates produced obvious changes. Two mutants were colony growth than the wild strain, the delta OL_s00043g381 mutant in PDA medium 1-6 growth rate of D at 24%-88% range, a AOL_s00078g577 mutant of 1-5 in the culture medium of D increased 11-22% in YMA; but a AOL_s00043g381 mutant of aerial mycelium than wild strain, and a AOL_s00078g577 mutant and wild-type strains compared to the mycelial growth was luxuriant; in conidiation, Delta AOL__s00043g381 mutant than wild strains reduced about 2.5 times, and a AOL_s00078g577 mutant sporulation increased 5.6 times: in the trap number, a AOL_s00078g577 in the 12,18,24 and 36 h, compared with the wild type strains were higher than 13,1 4,2.3 and 3 times: in nematicidal ability, AOL_s00078g577 mutant and wild-type strains than in 12,18 and 24 h, respectively higher than 60,9.6 and 2.1 times. The other mutant compared with wild strains in morphology, no significant differences in nematode trapping ability. Analysis of metabolites, compared to the HPLC atlas and fermentation liquid the crude extract of wild strains, type PKS gene AOL_s00043g828 Mutation Strains of isopentenyl and indole biosynthesis gene cluster transferase gene AOL_s00043g400 mutant, there are 6 peaks in the area increases, the area increased the peak in the delta AOL_s00043g828 mutant in 4, a AOL_s00043g400 mutant in 3 in addition, in the delta AOL_s00043g400 mutant and 5 peak area decreased, the 4 peaks disappeared.GC-MS results showed that AOL_s00043g828 mutant strains in the delta compound rich than the wild-type strain in specific mutation strains Compound 7, content increased the peak of 3, while only 3 wild strains in specific compounds. In the AOL_s00043g400 mutant, found in endemic wild strains in 7 compounds with 5 compounds containing isopentenyl units, in line with only 1 compounds containing isopentenyl units and 1 amide compounds in the mutant strain found in 1 increased the content of compound amino acids. At the same time that the P450 gene AOL_s00043g381, AOL_s00097g384 mutant compared with the wild strain, the change is mainly long chain aliphatic alkanes and long chain fatty esters. In addition, III the AOL_s00043g287 mutant type PKS gene, P450 gene and AOL_s00078g577 mutant strain AOL_s00097g233 fermentation broth extracts of HPLC and GC-MS were compared with the wild-type strain. There were no differences in gene expression profile analysis showed that the type I PKS gene AOL_s00043g 828 mutant gene expression have 151 significant differences, 94 downregulated genes, 57 up-regulated genes in P450 mutant strains, the AOL_s00097g384 mutation caused by the difference in gene expression was far more than a AOL_s00043g828 mutant, a total of 1231 genes expressed significant difference, expression of 743 genes were up-regulated 488 genes. According to the above experimental results, 7 genes in biosynthesis of this thesis, the gene type affects the metabolites of type I PKS gene, isopentenyl transferase gene and P450 gene, and the influence on the nematode trapping ability is 4 P450 in the P450 gene. Genes, AOL_s00043g381 and AOL_s00078g577 can influence the colony growth and sporulation, especially AOL_s00078g577 has significant effect on trap formation and nematode predation rate in AOL_s00043g381 and AOL_s00097g384 on metabolism The product has a certain influence, and knockout of AOL_s00097g384 caused the expression of 1231 genes changed. These results showed that P450 gene plays an important role in the growth and metabolism of a.oligosporus strains, and for the first time that P450 gene can regulate nematode trapping fungi trap formation and nematode trapping ability. In addition, this study suggested that in Arthrobotrys oligospora Baozhong, isopentenyl isopentenyl transferase gene AOL_s00043g400 may be involved in the biosynthesis of indole compounds in the transfer process, type III PKS gene AOL_s00043g287 and P450 gene AOL_s00043g287 may be in a state of silence under the experimental conditions. In conclusion, in Arthrobotrys oligospora Baozhong that part of biosynthetic genes of fungal phenotype, affect predation ability and secondary metabolites, but these biosynthetic genes is how to play the role of secondary metabolic production Between the phenotype and nematode and what relationship there is not clear, need to be further studied. This paper provided the strains for the secondary metabolism of product diversity and genetic diversity, which lays a foundation for further research of Arthrobotrys Baozhong less biosynthetic genes.

【学位授予单位】：云南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S476

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