發(fā)布時(shí)間：2018-01-20 08:54

  本文關(guān)鍵詞： 少孢節(jié)叢孢 生物合成基因 次級(jí)代謝產(chǎn)物 表型 捕食線蟲能力　出處：《云南大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：少孢節(jié)叢孢(Arthrobotrys oligospora)能產(chǎn)生豐富的次級(jí)代謝產(chǎn)物,但目前對(duì)其次級(jí)代謝產(chǎn)物生物合成基因的研究卻甚少。本論文根據(jù)少孢節(jié)叢孢中全基因組的生物合成基因的分析預(yù)測(cè)結(jié)果,篩選出7個(gè)生物合成基因作為研究對(duì)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異戊烯基轉(zhuǎn)移酶基因AOL_s00043g400, P450基因AOLs00043g381、AOL_s000789577.AOL_s00097g233.AOL_s00097g384。利用遺傳轉(zhuǎn)化手段,獲得了這7個(gè)基因的突變菌株,并對(duì)7個(gè)基因的突變株和野生菌株在菌落形態(tài)、菌落直徑生長(zhǎng)、產(chǎn)孢量、捕食線蟲能力及次級(jí)代謝產(chǎn)物等方面進(jìn)行了比較,此外,還比較了其中2個(gè)基因的突變株和野生菌株的基因表達(dá)差異。表型比較發(fā)現(xiàn),P450基因AOL_s00043g381和AOL_s00078g577突變株與野生菌株相比產(chǎn)生了較明顯的變化。兩株突變菌株菌落生長(zhǎng)均比野生菌株快,其中ΔOL_s00043g381突變株在PDA培養(yǎng)基中1-6 d增長(zhǎng)幅度達(dá)到了24%-88%不等,ΔAOL_s00078g577突變株在YMA培養(yǎng)基中1-5 d提高了11-22%；但ΔAOL_s00043g381突變株氣生菌絲比野生菌株少,而ΔAOL_s00078g577突變株與野生菌株相比菌絲生長(zhǎng)明顯茂盛；在產(chǎn)孢量上,ΔAOL__s00043g381突變株比野生菌株減少了約2.5倍,而ΔAOL_s00078g577突變株產(chǎn)孢量增加了5.6倍：在捕器數(shù)量上,ΔAOL_s00078g577在12,18,24和36 h,與野生菌株相比分別高出13,14,2.3和3倍：在殺線蟲能力上,ΔAOL_s00078g577突變株與野生菌株相比在12,18和24 h,分別高出60,9.6和2.1倍。其他基因突變株與野生菌株相比,在形態(tài),捕食線蟲能力上無顯著差異。代謝產(chǎn)物分析發(fā)現(xiàn),與野生菌株發(fā)酵液粗提物的HPLC圖譜相比,Ⅰ型PKS基因AOL_s00043g828突變株和吲哚生物合成基因簇上的異戊烯基轉(zhuǎn)移酶基因AOL_s00043g400突變株,均有6個(gè)峰的面積增大,其中面積明顯增高的峰在ΔAOL_s00043g828突變株中有4個(gè),ΔAOL_s00043g400突變株中有3個(gè), 此外,在ΔAOL_s00043g400突變株中還有5個(gè)峰面積減小,4個(gè)峰消失。GC-MS檢測(cè)結(jié)果顯示,ΔAOL_s00043g828突變菌株中的化合物要比野生菌株豐富,突變株中特有的化合物有7個(gè),含量明顯增高的峰有3個(gè),而野生菌株中只有3個(gè)特有化合物。在ΔAOL_s00043g400突變株中,發(fā)現(xiàn)在野生菌株中特有7個(gè)化合物中有5個(gè)為含有異戊烯基單位的化合物,在變株中僅有1個(gè)含異戊烯基單位的化合物和1個(gè)酰胺類化合物,在突變株中發(fā)現(xiàn)含量增加化合物中有1個(gè)氨基酸類化合物。同時(shí)發(fā)現(xiàn)P450基因ΔAOL_s00043g381、AOL_s00097g384突變株與野生菌株相比,發(fā)生變化的主要是長(zhǎng)鏈脂肪烷烴和長(zhǎng)鏈脂肪酯類化合物。此外,Ⅲ型PKS基因AOL_s00043g287突變株,P450基因AOL_s00078g577和AOL_s00097g233突變株發(fā)酵液粗提物的HPLC及GC-MS圖譜與野生菌株相比均無差異�；虮磉_(dá)譜分析發(fā)現(xiàn)Ⅰ型PKS基因AOL_s00043g828突變株有151個(gè)顯著差異表達(dá)基因,表達(dá)下調(diào)基因94個(gè),表達(dá)上調(diào)基因57個(gè)。在P450基因突變菌株中,AOL_s00097g384突變株中引起的差異表達(dá)基因遠(yuǎn)遠(yuǎn)多于ΔAOL_s00043g828突變株,共有1231個(gè)顯著差異表達(dá)基因,表達(dá)下調(diào)基因有743個(gè),表達(dá)上調(diào)基因488個(gè)。根據(jù)上述實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),在本論文研究的7個(gè)生物合成基因中,對(duì)代謝產(chǎn)物有影響的基因類型有Ⅰ型PKS基因、異戊烯基轉(zhuǎn)移酶基因和P450基因,對(duì)表型和捕食線蟲能力產(chǎn)生影響的都是P450基因。在4個(gè)P450基因中,AOL_s00043g381和AOL_s00078g577能影響菌落生長(zhǎng)及產(chǎn)孢,尤其是AOL_s00078g577對(duì)捕器形成和線蟲捕食率有顯著影響,此外,ΔAOL_s00043g381和AOL_s00097g384對(duì)代謝產(chǎn)物產(chǎn)生了一定影響,且AOL_s00097g384基因的敲除引起了1231個(gè)基因表達(dá)發(fā)生了顯著性變化。以上結(jié)果表明P450基因?qū)ι冁吖?jié)叢孢菌株生長(zhǎng)發(fā)育及代謝有重要作用,且首次發(fā)現(xiàn)P450基因能參與捕食線蟲真菌捕器形成及捕食線蟲能力的調(diào)控。此外,根據(jù)研究結(jié)果推測(cè),在少孢節(jié)叢孢中,異戊烯基轉(zhuǎn)移酶基因AOL_s00043g400可能參與吲哚類化合物生物合成的異戊烯基轉(zhuǎn)移過程,Ⅲ型PKS基因AOL_s00043g287及P450基因AOL_s00043g287在本實(shí)驗(yàn)條件下可能處于沉默狀態(tài)�？傊�,在少孢節(jié)叢孢中,部分生物合成基因能對(duì)真菌表型、捕食能力及次級(jí)代謝產(chǎn)物產(chǎn)生影響,但是這些生物合成基因是如何發(fā)揮作用,次級(jí)代謝產(chǎn)物與表型及捕食線蟲之間存在何種聯(lián)系還不清楚,有待進(jìn)一步探究。本論文為研究次級(jí)代謝產(chǎn)物多樣性及基因多樣性提供了菌株,為進(jìn)一步開展少孢節(jié)叢孢中生物合成基因的研究奠定了基礎(chǔ)。
[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.

【學(xué)位授予單位】：云南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S476

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相關(guān)期刊論文 前1條

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本文編號(hào)：1447618