海洋曲霉屬真菌次級(jí)代謝產(chǎn)物生物合成基因簇的基因組挖掘與遺傳轉(zhuǎn)化體系構(gòu)建
發(fā)布時(shí)間：2018-12-12 17:33
【摘要】：海洋微生物來源的天然產(chǎn)物因其獨(dú)特的結(jié)構(gòu)與生物學(xué)活性而備受關(guān)注,特別是有研究證實(shí)海洋動(dòng)植物體內(nèi)的微生物是其活性化合物的真正生產(chǎn)者。海蟑螂(Ligiaoceanic)是中國南方沿海漁民治療跌打損傷常用的藥用動(dòng)物,但目前對海蟑螂及其微生物的次級(jí)代謝產(chǎn)物(Secondary Metabolites,SMs)的研究較少,難以確認(rèn)其藥用活性成分。本研究以海蟑螂腸道內(nèi)海洋真菌Z5為研究對象,經(jīng)rDNA序列鑒定為曲霉屬菌株(Aspergillussp.)。應(yīng)用二代高通量測序技術(shù)對該菌株進(jìn)行全基因組測序分析及antiSMASH基因簇分析,結(jié)果顯示Z5菌株基因組大小為33.8 Mbp,系統(tǒng)進(jìn)化處于A.versicolor分支,共含有89個(gè)SMs生合成基因簇,結(jié)合One-Strain-Many-Compounds(OSMAC)結(jié)果說明:該菌具有合成多種SMs的潛能;全基因組挖掘的SMs基因簇?cái)?shù)量遠(yuǎn)超OSMAC分析的化合物數(shù)量,多數(shù)基因簇在現(xiàn)有培養(yǎng)條件下為沉默狀態(tài),如激活這些沉默基因簇的表達(dá),極可能獲得新型活性化合物。為開展該菌株的生物合成研究而獲得新型化合物,本研究構(gòu)建了 Z5的遺傳轉(zhuǎn)化系統(tǒng),探索優(yōu)化原生質(zhì)體制備純化方法,確定可通過β-巰基乙醇預(yù)處理及蔗糖分層純化法獲得再生率為94%的高質(zhì)量原生質(zhì)體,并應(yīng)用Double-Joint PCR替代傳統(tǒng)酶切連接法構(gòu)建基因盒,最終通過PEG/CaCl2轉(zhuǎn)化,獲得Z5的營養(yǎng)缺陷型菌株。本研究為海洋真菌新型天然產(chǎn)物的獲得提供重要來源,為開發(fā)應(yīng)用該類真菌天然產(chǎn)物奠定基礎(chǔ),有助于闡明海洋真菌系統(tǒng)進(jìn)化及與海洋動(dòng)植物活性產(chǎn)物的關(guān)聯(lián),為開發(fā)利用其它海洋真菌活性天然產(chǎn)物提供經(jīng)驗(yàn)方法。
[Abstract]:The natural products derived from marine microorganisms have attracted much attention because of their unique structure and biological activities. Especially, some studies have confirmed that the microorganisms in marine animals and plants are the real producers of their active compounds. Sea cockroach (Ligiaoceanic) is a common medicinal animal used by fishermen in southern China to treat the damage. However, there are few studies on the secondary metabolites (Secondary Metabolites,SMs) of sea cockroaches and their microbes, so it is difficult to confirm the medicinal active components of sea cockroaches. In this study, the marine fungus Z5 in the intestinal tract of sea cockroach was identified as (Aspergillussp.). By rDNA sequence analysis. The second generation high-throughput sequencing technique was used to analyze the whole genome and the antiSMASH gene cluster of the strain. The results showed that the genome size of Z5 strain was 33.8 Mbp, phylogeny in the A.versicolor branch, and there were 89 SMs synthetic gene clusters. The results of One-Strain-Many-Compounds (OSMAC) showed that the strain had the potential to synthesize multiple SMs. The number of SMs gene clusters mined by the whole genome is much larger than the number of compounds analyzed by OSMAC. Most of the gene clusters are silenced under the existing culture conditions. If the expression of these silent gene clusters is activated, it is very possible to obtain new active compounds. In order to study the biosynthesis of the strain and obtain new compounds, the genetic transformation system of Z5 was constructed to optimize the preparation and purification of protoplasts. High quality protoplasts with regeneration rate of 94% could be obtained by 尾 -mercaptoethanol pretreatment and sucrose stratification purification. The gene cassette was constructed by Double-Joint PCR instead of traditional enzyme ligation method, and finally transformed by PEG/CaCl2. A nutrient deficient strain of Z5 was obtained. This study provides an important source for the acquisition of new natural products of marine fungi, lays a foundation for the development and application of these natural products, and helps to clarify the phylogeny of marine fungi and their association with the active products of marine flora and fauna. To provide an empirical method for the development and utilization of other marine fungi active natural products.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q933
本文編號(hào)：2375003
[Abstract]:The natural products derived from marine microorganisms have attracted much attention because of their unique structure and biological activities. Especially, some studies have confirmed that the microorganisms in marine animals and plants are the real producers of their active compounds. Sea cockroach (Ligiaoceanic) is a common medicinal animal used by fishermen in southern China to treat the damage. However, there are few studies on the secondary metabolites (Secondary Metabolites,SMs) of sea cockroaches and their microbes, so it is difficult to confirm the medicinal active components of sea cockroaches. In this study, the marine fungus Z5 in the intestinal tract of sea cockroach was identified as (Aspergillussp.). By rDNA sequence analysis. The second generation high-throughput sequencing technique was used to analyze the whole genome and the antiSMASH gene cluster of the strain. The results showed that the genome size of Z5 strain was 33.8 Mbp, phylogeny in the A.versicolor branch, and there were 89 SMs synthetic gene clusters. The results of One-Strain-Many-Compounds (OSMAC) showed that the strain had the potential to synthesize multiple SMs. The number of SMs gene clusters mined by the whole genome is much larger than the number of compounds analyzed by OSMAC. Most of the gene clusters are silenced under the existing culture conditions. If the expression of these silent gene clusters is activated, it is very possible to obtain new active compounds. In order to study the biosynthesis of the strain and obtain new compounds, the genetic transformation system of Z5 was constructed to optimize the preparation and purification of protoplasts. High quality protoplasts with regeneration rate of 94% could be obtained by 尾 -mercaptoethanol pretreatment and sucrose stratification purification. The gene cassette was constructed by Double-Joint PCR instead of traditional enzyme ligation method, and finally transformed by PEG/CaCl2. A nutrient deficient strain of Z5 was obtained. This study provides an important source for the acquisition of new natural products of marine fungi, lays a foundation for the development and application of these natural products, and helps to clarify the phylogeny of marine fungi and their association with the active products of marine flora and fauna. To provide an empirical method for the development and utilization of other marine fungi active natural products.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q933
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