【摘要】：生防鏈霉菌Act12是從自青藏高原分離獲得的一株多效放線菌,前期的科學(xué)研究表明該菌株具有多種良好的生物活性,例如抑制病原真菌的生長(zhǎng),促進(jìn)草莓、甜瓜和棉花等多種農(nóng)作物的生長(zhǎng)等。通過(guò)對(duì)該菌株進(jìn)行全基因組測(cè)序并提交antiSMASH在線分析發(fā)現(xiàn)Act12基因組中可能含有豐富的次級(jí)代謝產(chǎn)物生物合成基因簇,然而,對(duì)實(shí)驗(yàn)室條件下Act12的發(fā)酵液進(jìn)行檢測(cè)發(fā)現(xiàn)該菌株含有的次級(jí)代謝產(chǎn)物的含量及種類(lèi)都較少,這表明Act12中絕大多數(shù)基因簇是沉默的或低表達(dá)的。為了激活A(yù)ct12中的沉默基因簇,挖掘該菌株中可能的活性產(chǎn)物資源,并進(jìn)一步探究相關(guān)的活性機(jī)理,我們通過(guò)同源重組的方法構(gòu)建了Act12的四株可穩(wěn)定遺傳的突變株,包括可能的LuxR家族調(diào)控因子SPA4754的缺失突變株Δspa4754、可能的TetR家族調(diào)控因子SPA0520的缺失突變株Δspa0520、全局性調(diào)控基因nsdA的缺失突變株ΔnsdA和全局性調(diào)控基因bldA的缺失突變株ΔbldA。通過(guò)病原微生物拮抗活性實(shí)驗(yàn)以及比較代謝圖譜學(xué)分析后發(fā)現(xiàn):缺失突變菌株Δspa0520對(duì)幾類(lèi)病原供試菌的抑制活性較野生型菌株Act12的活性顯著增強(qiáng)。HPLC的代謝圖譜學(xué)分析結(jié)果表明,突變株Δspa0520的發(fā)酵產(chǎn)物中較野生型Act12出現(xiàn)一顯著差異峰。對(duì)突變株Δspa0520進(jìn)行大量發(fā)酵,純化顯著差異峰所對(duì)應(yīng)的化合物,通過(guò)高分辨質(zhì)譜分析等手段對(duì)目的化合物進(jìn)行初步的結(jié)構(gòu)分析及鑒定,最終分析得出該化合物為寡霉素D。同時(shí),對(duì)突變株Δspa0520進(jìn)行遺傳互補(bǔ)實(shí)驗(yàn)獲得相應(yīng)的遺傳互補(bǔ)菌株Δspa0520C,并檢測(cè)其對(duì)病原微生物拮抗活性以及比較代謝圖譜學(xué)分析,結(jié)果發(fā)現(xiàn):遺傳互補(bǔ)菌株Δspa0520C對(duì)幾類(lèi)病原供試菌的抑制活性及其HPLC代謝圖譜與野生型Act12保持一致。缺失突變菌株Δspa0520的相關(guān)實(shí)驗(yàn)結(jié)果表明spa0520(GenBank Accession No.KY368145)可能是寡霉素D生物合成途徑中的負(fù)調(diào)控基因,也可能具有多效調(diào)控作用,這為研究有關(guān)鏈霉菌Act12的其它次級(jí)代謝產(chǎn)物的調(diào)控機(jī)理提供了幫助,同時(shí)采用敲除負(fù)調(diào)控基因的方法為激活相關(guān)的沉默基因簇提供了有益的借鑒。
[Abstract]:Biocontrol Streptomyces Act12 is a multifunctional actinomycetes isolated from the Qinghai-Tibet Plateau. Previous scientific studies have shown that the strain has many good biological activities, such as inhibiting the growth of pathogenic fungi and promoting strawberry. The growth of a variety of crops, such as melon and cotton. By sequencing the whole genome of the strain and submitting it to antiSMASH online analysis, it was found that the Act12 genome may contain abundant secondary metabolites biosynthesis gene clusters, however, The detection of the fermentation broth of Act12 in laboratory showed that there were few secondary metabolites in this strain, which indicated that the majority of gene clusters in Act12 were silent or low expressed. In order to activate the silencing gene cluster in Act12, excavate the possible active product resources in the strain, and further explore the related activity mechanism, we constructed four stable genetic mutants of Act12 by homologous recombination. The deletion mutant 螖 spa4754, of the possible LuxR family regulator SPA4754, the deletion mutant 螖 spa0520, of the TetR family regulator SPA0520, the deletion mutant 螖 nsdA of the global regulatory gene nsdA and the deletion mutant 螖 bldA. of the global regulatory gene bldA The antagonistic activity of pathogenic microorganisms and the comparative analysis of metabolic atlas showed that the inhibitory activity of deletion mutant 螖 spa0520 was significantly higher than that of wild-type strain Act12. The results of metabolic atlas of HPLC showed that the inhibitory activity of deletion mutant 螖 spa0520 was significantly higher than that of wild-type strain Act12. There was a significant difference peak in the fermentation products of the mutant 螖 spa0520 compared with the wild-type Act12. The mutant 螖 spa0520 was fermented in large quantities, and the compounds corresponding to the significant difference peak were purified. The target compounds were analyzed and identified by means of high resolution mass spectrometry. Finally, the compounds were identified as oligomycin D. At the same time, the genetic complementary strain 螖 spa0520C, was obtained by genetic complementation test on the mutant 螖 spa0520, and its antagonistic activity against pathogenic microorganisms was detected and the comparative metabolic map analysis was carried out. The results showed that the inhibitory activity of genetic complementary strain 螖 spa0520C and its HPLC metabolic map were consistent with those of wild type Act12. The experimental results of deletion mutant 螖 spa0520 suggest that spa0520 (GenBank Accession No.KY368145 may be a negative regulation gene in the biosynthesis pathway of oligomycin D, and may also have multiple effects. It is helpful to study the regulation mechanism of other secondary metabolites of Streptomyces Act12, and to use the method of knockout of negative regulatory genes to activate the related silencing gene clusters.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S476.1;Q78

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