【作者】 杨倩；

【导师】 梅艳珍； 戴传超；

【作者基本信息】 南京师范大学 ， 微生物学， 2020， 硕士

【摘要】 黄酮类物质是天然的芳香类次生代谢产物,通常与植物的抗病性能、花色形成及生长发育等密切有关。前期课题组从大戟科重阳木(Bischofia polycarpa)的茎内皮中分离出一株内生真菌枫香拟茎点霉(Phomopsis liquidambaris,B3),研究表明该菌株可与花生(Arachis hypogaea)、水稻(Oryza sativa L.)等建立共生体系。B3接种水稻后,可在低氮条件下促进水稻对氮素的吸收,加快水稻的生长;B3与其他固氮结瘤菌一起协同定殖花生,可促进花生结瘤,缓解花生连作障碍,提高花生产量。基于内生真菌B3的生态作用,通过对B3基因组的全测序及次级代谢产物信息分析发现,内生真菌B3具有黄酮类物质合成相关基因簇,且前期的研究发现采用CRISPR/Cas9靶向基因敲除系统敲除B3中的全局性调控因子丝裂原活化蛋白激酶激酶(mapkk)基因后,该突变菌株在马铃薯葡萄糖液体培养基(PDB)的发酵过程中可产生类似于黄酮类物质的黄色物质。然而,在野生型B3中黄酮类物质的合成量较少或几乎检测不到。研究发现,Δmapkk菌株黄酮类物质的生成比野生型显著提高,本研究旨在探究内生真菌B3产生黄酮类物质的合成规律,分析合成关键基因和调控规律,为黄酮类物质合成提供可能的宿主及为农业抗病奠定基础。首先,我们以Δmapkk菌株为原始菌株,利用高效液相色谱及质谱联用的技术对菌株的发酵产物进行了鉴定,结果显示Δmapkk菌株可产生柚皮素、槲皮素及山奈酚等黄酮类物质,这说明内生真菌B3可合成黄酮类物质。随后,对Δmapkk菌株产关键产物柚皮素的发酵条件进行了发酵时间、葡萄糖浓度及发酵方式等方面的单因素分析,结果表明,Δmapkk菌株在发酵36 h时,其柚皮素产量最高(126.73±5.95μg/g);发酵液中的葡萄糖浓度为20 g/L时,其产量最高(113.35±1.36μg/g);发酵方式为补料分批发酵而不是批次发酵时,其产量最高(186.35±10.25μg/g)。其次,利用基因组装技术构建了丝状真菌表达载体,并采用此方法快速构建了丝状真菌表达载体用于筛选B3内源性的强启动子,结果表明,启动子序列GPDp、ADHp、PGIp(500 bp)、PGKp(700 bp)及Unigene16948p(366 bp)可高效表达m Cherry信号基因,可用于后续基因的过表达。本研究将筛选的启动子用于黄酮合成关键酶查尔酮合成酶和查尔酮异构酶的表达,对B3菌中黄酮类物质的合成进行了正向的促进,使得OE-CHS1-CHI和OE-CHS2-CHI过表达菌株分别产生42.7±2.1μg/L及44.6±1.58μg/L的柚皮素。接着采用CRISPR/Cas9靶向基因敲除系统成功敲除了内生真菌B3中的CHS基因(CHS1、CHS2),探究其生理生化特性时发现CHS基因的敲除可抑制菌丝的向外延伸,影响了菌株细胞壁的形成。为了探究内生真菌B3中CHS基因的过表达对花生生长的影响,分别将野生型B3、过表达菌株(OE-CHS1-CHI、OE-CHS2-CHI)及ΔCHS1CHS2接种花生,结果表明,内生真菌B3中CHS基因的过表达在一定程度上促进了花生的生长。最后,我们对内生真菌B3中查尔酮合成酶基因进行了研究,从序列角度分析发现内生真菌B3中含有2条查尔酮合成酶基因(命名为CHS1、CHS2),CHS1基因的全长为1365 bp,含有一个长度为126 bp的内含子,CHS2的基因全长为483 bp,不含内含子序列,其中CHS1基因的序列特征与大多数植物来源的查尔酮合酶基因更为相似,且CHS1蛋白序列与其他物种的序列相似度高于CHS2,具有更多的保守区域。从结构角度分析发现CHS1和CHS2蛋白均不含有蛋白跨膜结构域及信号肽序列。综上所述,内生真菌B3具有柚皮素及其下游黄酮类物质的合成途径,且mapkk基因的敲除可激活该途径。同时通过对内生真菌B3中黄酮合成相关基因的研究,对自身的代谢进行了正向调控,说明黄酮对植物的生长发育具有重要的价值。因此,本研究对快速生产有价值的黄酮类物质柚皮素、槲皮素及山奈酚等具有重要的参考意义以及对农业的促生及防御病虫害等方面具有一定指导作用。更多还原

【Abstract】 Flavonoids are natural aromatic secondary metabolites,which are usually closely related to the disease resistance,flower color formation and growth of plants.The previous research team isolated a broad-spectrum endophytic fungus Phomopsis liquidambaris(B3)from the inner bark of Bischofia polycarpa.Studies have shown that the endophytic fungus B3 can establish a symbiotic system with rice(Oryza sativa L.)and peanuts(Arachis hypogaea)etc.After B3 is inoculated with rice,it can promote the absorption of nitrogen by rice and accelerate the growth of rice under low nitrogen conditions;B3 and other nitrogen-fixing nodule bacteria cooperatively colonize peanuts can promote peanut nodulation,relieve peanut continuous cropping obstacles,and increase peanut yield.Based on the ecological function of endophytic fungus B3,through complete sequencing of the B3 genome and information analysis of secondary metabolites,it was found that the endophytic fungus B3 has gene clusters related to the synthesis of flavonoids.Previous studies found that the CRISPR/Cas9 targeted gene knockout system knocked out the global regulatory factor mapkk(mitogen activated protein kinase kinase)gene in B3,the mutant strain can produce yellow substances similar to flavonoids during the fermentation of potato glucose liquid medium(PDB).However,in wild type B3,the amount of flavonoids synthesis is small or almost undetectable.It was found that the production of flavonoids inΔmapkk was significantly higher than that in the wild type.The purpose of this study was to explore the synthesis rule of flavonoids produced by endophytic fungus B3,analyze the synthesis key genes and regulation rule,and provide a possible host for the synthesis of flavonoids and lay a foundation for agricultural disease resistance.First,we useΔmapkk strains as the original strains,using high performance liquid chromatography(HPLC)and mass spectrometry technology the identification of the strains of fermentation product,the results showedΔmapkk strains can produce naringenin,quercetin and kaempferol.This suggests that the endophytic fungus B3 has the ability to synthesize flavonoids.Subsequently,a single factor analysis of fermentation time,glucose concentration and fermentation method was conducted on the fermentation conditions of the key product naringenin produced by theΔmapkk strain.Then,the single factor analysis of fermentation conditions,such as fermentation time,glucose concentration and fermentation mode was carried out.The results showed that the highest yield of naringenin was(126.73±5.95μg/g)when the fermentation time was 36 hours,and the highest yield was(113.35±1.36μg/g)when the glucose concentration was 20 g/L.The highest yield(186.35±10.25μg/g)was obtained when fed batch fermentation was used instead of batch fermentation.Secondly,the filamentous fungal expression vector was constructed using gene assembly technology,and the filamentous fungal expression vector was quickly constructed using this method for screening B3 endogenous strong promoter.The results showed that 500 bp promoter sequences GPDp,ADHp,PGIp and 700 bp PGKp and 366 bp Unigene16948p could efficiently express m Cherry signal gene and could be used for over expression of subsequent genes.In this study,the selected promoter was used for the expression of chalcone synthase and chalcone isomerase,which promoted the synthesis of flavonoids in B3 strain,and the overexpression strain OE-CHS1-CHI and OE-CHS2-CHI could produce 42.7±2.1μg/L and44.6±1.58μg/L naringenin.Then,CRISPR/cas9 targeted gene knockout system was used to knock out CHS gene(CHS1,CHS2)in endophytic fungus B3 successfully.When exploring its physiological and biochemical characteristics,it was found that the knockout of CHS gene could inhibit the outward extension of mycelium and affect the formation of cell wall of strain.In order to investigate the effect of overexpression of CHS gene in endophytic fungus B3 on peanut growth,wild type B3,overexpression strains(OE-CHS1-CHI,OE-CHS2-CHI)andΔCHS1CHS2 was inoculated with peanuts.The results showed that the overexpression of CHS gene in endophytic fungus B3 promoted the growth of peanuts to a certain extent.Finally,we studied the gene of chalcone synthase in endophytic fungus B3,and found that there were two genes of chalcone synthase(named CHS1 and CHS2)in endophytic fungus B3.The total length of CHS1 gene was 1365 bp,and there was an intron with a length of 126 bp,while the total length of CHS2 gene was 483 bp does not contain intron sequence.CHS1 gene is more similar to chalcone synthase gene from most plants.and the sequence similarity of the CHS1 protein sequence to other species is higher than that of CHS2,with more conserved regions.From a structural perspective,it was found that neither CHS1 nor CHS2 proteins contained protein transmembrane domains or signal peptide sequences.In conclusion,endophytic fungus B3 has a synthetic pathway of naringenin and its downstream flavonoids,and the knockout of mapkk gene can activate this pathway.At the same time,through the study of the genes related to flavonoids synthesis in endophyte B3,the metabolism of the endophyte B3 was positively regulated,which indicated that flavonoids have important value for plant growth and development.This study has important reference significance for the rapid production of valuable flavonoids,such as naringenin,quercetin and kaempferol,and has certain guiding role in promoting the growth of agriculture and preventing diseases and insect pests.更多还原