本文選題：少孢節(jié)叢孢　切入點：生物合成基因　出處：《云南大學》2016年碩士論文　論文類型：學位論文

【摘要】：本文以捕食線蟲真菌的模式菌株少孢節(jié)叢孢(Arthrobotrys oligospora)中的1個Ⅰ型聚酮合酶(PKS)基因AOL_s00079g496、1個萜類合成酶(TPS)基因AOL_s00079g224及3個細胞色素P450基因AOL_s00079g225、AOL_s00043g740和AOL_s00210g57為研究對象,通過敲除質粒構建和原生質體轉化方法獲得了6個敲除突變菌株。通過對這6株突變菌株與野生菌株在菌絲形態(tài)、菌絲生長直徑、產孢量、孢子萌發(fā)、三維菌網生成和捕食線蟲能力及其次生代謝產物圖譜等方面比較分析,初步探究了這些生物合成基因對少孢節(jié)叢孢次生代謝產物圖譜及其形態(tài)、侵染能力的影響。表型比較結果顯示,這6株突變株與野生菌株均有不同程度的差異,其中PKS基因AOL_s00079g496-KS和兩個P450基因AOL_s00079g225和AOL_s00210g57的突變株表型差異較為突出,在菌絲形態(tài)上,與野生菌株相比這三株突變菌的菌絲明顯茂盛,其中突變株MOL_s00079g496-KS最為顯著,其它菌株則無明顯差異。在菌絲生長直徑上,與野生菌株相比PKS基因突變株△AOL_s00079g496-KS及 △AOL_s00079g496-KR在富營養(yǎng)培養(yǎng)基TYGA平板上生長稍慢,另一株TPS基因突變株△AOL_s00079g224在乏營養(yǎng)培養(yǎng)基CMA平板上菌絲生長直徑也明顯小于野生菌株,P450基因突變株△AOL_s00079g225、AAOL_s00210g57及△AOL_s00043g740則無顯著差異。在產孢量上,除△AOL_s00210g57的產孢量與野生菌株無明顯差異外,其他突變株產孢量均高于野生菌株,增加范圍為58%-300%,其中突變株△AOL_s00079g224最為顯著。在孢子萌發(fā)上,所有突變株相比野生菌株都有不同程度的減緩,2h時表現(xiàn)最為明顯,不同突變菌株的孢子萌發(fā)率的減少幅度為24%-70%,其中TPS基因突變株△AOL_s00079g224最為顯著。在捕器數量上,P450基因突變株△AOL_s00079g225與野生菌株的捕器數量差異最為顯著,在12 h、24 h、36 h分別增加了約49%、35%、20%,其他突變菌株則無顯著性差異。在捕食線蟲能力上,與野生菌株相比有顯著差異的有PKS基因突變株△AOL_s00079g496-KS、 △AOL_s00079g496-KR及P450基因突變株△AOL_s00079g225、△AOL_s002I0g57,其中突變株△AOL_s00079g496-KS、△AOL_s00079g496-KR在24 h時線蟲捕食率明顯低于野生菌株,分別降低了約41%和32%；而突變株△AOL_s00079g225與△AOL_s00210g57在12h時線蟲捕食率比野生菌株提高了約2倍和3倍,其他突變株則無顯著性差異。HPLC檢測圖譜比較結果顯示,Ⅰ型PKS基因AOL_s0079g496的KS結構域敲除后對少孢節(jié)叢孢次生代謝產物的影響變化最大,突變株△AOL_s00079g496-KS與野生菌株相比至少缺失了5個峰,新增了8個峰,并且至少有4個峰面積發(fā)生了變化；3個P450基因的突變株與野生菌株相比變化的主要是峰面積,其中△AOL_s00079g225峰面積發(fā)生變化的峰有7個,△AOL_s00210g57及△AOL_s00043g740峰面積發(fā)生變化的峰有11個。GC-MS檢測結果顯示,AOL_s00079g225敲除后有6個萜類化合物發(fā)生變化,因此推測P450基因AOL_s00079g225的功能可能是對TPS基因AOL_s00079g224的產物進行修飾；AOL_s00043g740敲除后,有9個脂肪烷類和5個直鏈萜類化合物發(fā)生了變化,而P450單加氧酶會參與不飽和脂肪酸的代謝及萜類的合成,因此推測P450基因AOL_s00043g740可能對不飽和脂肪酸及直鏈萜類化合物進行修飾；在PKS基因AOL_s00079g496、TPS基因AOL_s00079g224及P450基因AOL_s000210g57的突變株中沒有檢測到哪一類化合物明顯發(fā)生變化,因此它們具體參與哪些化合物的合成還有待進一步研究。值得注意的是,發(fā)現(xiàn)在三株P450基因的突變株發(fā)酵液中都檢測到了5-methyl-2-((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)be-nzene-1,3-diol這個化合物,它與少孢節(jié)叢孢中已經鑒定的節(jié)叢孢素類化合物有相似的骨架結構,因此推測這三個P450基因參與的合成途徑可能與這類化合物相關。本論文研究發(fā)現(xiàn),在五個三種不同類型的生物合成基因中,對形態(tài)和捕食能力影響較大的基因的有Ⅰ型PKS基因AOL_s00079g496和P450基因AOL_s00079g225、 AOL_s00210g57,對次生代謝產物影響較大的為Ⅰ型PKS基因AOL_s00079g496和TPS基因AOL_s0079g224。其中PKS基因AOL_s00079g496敲除后少孢節(jié)叢孢在形態(tài)和次生代謝產物上都有較大變化,P450基因AOL_s00079g225與AOL_s00210g57敲除后對少孢節(jié)叢孢的影響則主要表現(xiàn)在形態(tài)上。以上結果表明,少孢節(jié)叢孢中部分生物合成基因與其形態(tài)及次生代謝組學相關聯(lián),本論文也是首次發(fā)現(xiàn)捕食線蟲真菌中的Ⅰ型PKS基因及P450基因能參與調控其菌絲形態(tài)、捕器形成及捕食線蟲能力。
[Abstract]:Based on the model strain of nematode trapping fungus a.oligosporus (Arthrobotrys oligospora) 1 type of polyketide synthase (PKS) gene AOL_s00079g496,1 terpene synthase (TPS) gene AOL_s00079g224 and 3 cytochrome P450 genes AOL_s00079g225, AOL_s00043g740 and AOL_s00210g57 as the research object, through the knockout plasmid and protoplast conversion method to take 6 knockout mutant strains. Based on these 6 mutant strains and wild strains in mycelial morphology, mycelial growth diameter, sporulation, spore germination, comparative analysis of 3D mesh generation and predator nematode bacteria ability and secondary metabolites of atlas, preliminary explore these biosynthetic genes for less Arthrobotrys secondary metabolites map and its morphology, affect the infection ability. The phenotypic comparison shows that the 6 mutant strains and wild strains had difference in different degrees, the In the PKS AOL_s00079g496-KS gene and two P450 genes AOL_s00079g225 and AOL_s00210g57 mutant phenotypes is more prominent in mycelial morphology, compared with wild strains of these three mutant strains of mycelium was lush, including the mutant MOL_s00079g496-KS was the most significant, and there were no significant differences in other strains. The diameter of mycelial growth, compared with wild strain the PKS mutant Delta AOL_s00079g496-KS and delta AOL_s00079g496-KR medium plate TYGA growth slightly slower in rich nutrition, another strain of TPS mutant AOL_s00079g224 CMA medium plate on the mycelial growth of smaller diameter than the wild strain in the lack of nutrition, the P450 mutant AOL_s00079g225, there is no significant difference between AAOL_s00210g57 and AOL_s00043g740. In sporulation, in addition to no significant difference between the sporulation of delta AOL_s00210g57 and wild strain and other mutant sporulation were Higher than that of the wild strain, increase the range of 58%-300%, the mutant AOL_s00079g224 was the most significant. In spore germination, all mutant wild strains had slowed to varying degrees, 2h was the most obvious, the germination rate of spores of different mutant strains reduced the amplitude of 24%-70%, the TPS gene mutant Delta AOL_s00079g224 significant. In trap number, P450 mutant AOL_s00079g225 and wild strain of the trap number difference was most significant at 12 h, 24 h, 36 h were increased by about 49%, 35%, 20%, other strains had no significant difference. The nematode trapping ability, compared with the wild there is a significant difference of strain PKS mutant Delta AOL_s00079g496-KS, Delta AOL_s00079g496-KR and P450 mutant Delta AOL_s00079g225, Delta AOL_s002I0g57, the mutant Delta AOL_s00079g496-KS, Delta AOL_s00079g496-KR at 24 h Nematode predation rate was significantly lower than that of the wild strain, were reduced by 41% and 32%; and delta AOL_s00079g225 and delta AOL_s00210g57 mutant in 12h nematode predation rate than wild strain increased by about 2 times and 3 times the other mutants had no significant difference.HPLC detection map comparison results show that the KS domain of PKS the biggest change in AOL_s0079g496 gene knock on effect on a.oligosporus secondary metabolites of mutant AOL_s00079g496-KS, compared with the wild strain missing at least 5 peaks, 8 new peaks, and at least 4 were changed to a peak area; 3 P450 gene mutant compared with the wild strain changes is the main peak area of the delta AOL_s00079g225 peak area changes the peak of 7, Delta AOL_s00210g57 and delta AOL_s00043g740 peak area changes the peak 11.GC-MS test results showed that AOL_s00079g225 knockout There are 6 terpenoids change, suggesting that P450 gene AOL_s00079g225 may function is the product of TPS gene modified AOL_s00079g224; AOL_s00043g740 knockout, has changed 9 fat 5 straight chain alkanes and terpenes, and P450 monooxygenase in unsaturated metabolism and synthesis terpene fatty acids, suggesting that P450 gene AOL_s00043g740 may be of unsaturated fatty acid and straight chain terpenoids were modified; in the PKS AOL_s00079g496 gene, TPS gene mutation and P450 gene of AOL_s00079g224 AOL_s000210g57 were not detected in which compounds have changed obviously, so they are specifically involved in the synthesis of compounds which have yet to be further research. It is worth noting that the mutant found in the fermentation liquid of P450 genes of three strains were detected by 5-methyl-2- ((2E, 6E) -3,7,11-trimethyldodeca-2 6,10-trien-1-yl, be-nzene-1,3-diol) of this compound, it has Baosu Arthrobotrys compounds and identification of Arthrobotrys oligospora Baozhong has the skeleton structure similar, suggesting that the synthesis pathway of three P450 genes may participate with these compounds. This study found that in five of three different types of biosynthetic genes. The morphology and predation ability influence gene with type I PKS gene AOL_s00079g496 and P450 gene AOL_s00079g225, AOL_s00210g57, on the secondary metabolites of type I PKS gene AOL_s00079g496 and TPS gene AOL_s0079g224. PKS AOL_s00079g496 gene knockout a.oligosporus have great changes in morphology and secondary metabolites, P450 AOL_s00079g225 and AOL_s00210g57 gene knock of a.oligosporus after is mainly manifested in the form. The above results show that less spore Festival Learn the associated part of Baozhong plexus biosynthesis gene and its morphology and secondary metabolism group, this thesis is the first type of PKS gene and P450 gene in nematode trapping fungi can participate in the regulation of the mycelial morphology, trap formation and nematode trapping ability.

【學位授予單位】：云南大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S476

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