【作者】 刘柳；

【导师】 李斌；

【作者基本信息】 浙江大学 ， 植物病理学， 2020， 硕士

【摘要】 环L-苯丙氨酸L-脯氨酸(cyclo-(L-Pro-L-Phe),cFP)是一种非常重要的天然产物,已在多种细菌和真菌的次生代谢产物中被报道。国内外研究已发现cFP具有抗真菌、抗细菌、抗病毒和诱导群体感应等多种生物活性,这些优良特性使得cFP在植物病害的生物防治上有广阔的应用前景,此时,高效生物合成cFP就成为关键,然而目前关于cFP生物合成的调控机制仍未完全明确。最近国内有研究发现洋葱伯克氏菌(Burkholderia cepacia)CF-66能产生对半裸镰刀菌(Fusarium semitectum)菌丝生长有明显抑制效果的cFP。有趣的是,我们研究也发现实验室前期从稻田土壤分离获得的一株拮抗细菌Burkholderia seminalis R456及其代谢物能够显著抑制小麦赤霉病菌(Fusarium graminearum,Fg;禾谷镰刀菌)的菌丝生长,这些结果使得我们有理由推测cFP或许是拮抗细菌R456抑制Fg的活性物质之一。本研究基于这一假设,开展了以下工作:为搞清楚R456拮抗Fg是否至少部分由于分泌抗菌物质cFP,首先利用平板对峙法测定了实验室前期构建的菌株R456的997个Tn5插入突变体对Fg的拮抗效果,获得了拮抗效果增强(与野生型相比增强20%以上)的突变体8个(编号为88、189、117、113、885、495、268和255),拮抗效果减弱(与野生型相比减弱20%以上)的突变体5个(编号为216、123、85、224和225);然后利用高效液相色谱(HPLC)分析了上述13个拮抗效果显著改变的Tn5突变体的上清滤液,结果发现突变体88相对于其它突变体和野生型,在20 min的出峰时间点有一个明显的差异峰,且相同实验条件下该差异峰的出峰时间与人工合成的cFP标准品基本一致;接着对这个差异峰进行LC-MS分析,一级质谱图结果显示其相对分子质量为245.12,与cFP的相对分子质量接近,二级质谱图结果显示其共有7个碎片峰,相对分子质量分别为65.04、70.06、92.05、154.07、172.11、217.13、245.12,与cFP的化学结构接近。最后平板拮抗实验结果显示,菌株R456生物合成的cFP和人工合成的cFP都能强烈抑制Fg的菌丝生长,且拮抗效果在0.1-1000 ppm范围内随着浓度的增高而增强。为探究拮抗细菌R456抗菌物质cFP的合成调控机制,首先,我们通过反向PCR扩增上述13个拮抗效果改变突变体的插入位点基因序列,并将测序结果与R456全基因组进行比对分析,成功鉴定出突变体88插入位点所在基因为Dct转运系统【负责四碳二羧酸C4-dicarboxylates(dCAs)转运,主要成分为Dct-A,-B,-D】中的DctB。接着构建了Dct转运系统主要组份-A,-B,-D的基因敲除突变体和回补体,并利用HPLC测定其上清滤液中的cFP含量,结果显示,Dct(-A、-B和-D)突变体的cFP含量相比野生型有明显上升,但相应回补体的cFP产量与野生型无显著差异,显示R456的cFP合成与Dct转运系统密切相关。最后,利用HPLC分析了Dct转运系统底物dCAs对R456及其突变体中cFP产量的影响,实验结果显示,在野生型菌株中添加草酰乙酸、苹果酸、天冬氨酸和琥珀酸等dCAs,会导致其cFP产量与对照相比显著下降,而在Dct转运系统的3个突变体菌株(Dct-A、-B和-D)中添加上述四种dCAs,其cFP产量与对照相比无明显变化,说明dCAs能够负调控菌株R456中cFP的合成。综上所述,本研究清楚地显示cFP是菌株R456拮抗Fg的主要活性物质之一,其生物合成受到dCAs的负调控。更多还原

【Abstract】 Cyclic L-phenylalanine L-proline(cyclo-(L-pro-L-phe),cFP)is a very important natural product and has been reported in the secondary metabolites of a variety of bacteria and fungi.With cFP antifungal,antibacterial,antiviral and induction of quorum sensing and many other biological activities,cFP has become the research focus in biological control of plant diseases field.From the application prospects,the effective biological synthetic of cFP is of importance,however,the current regulatory mechanism about the biosynthesis of cFP is still not entirely clear.Recently,some domestic studies have found that Burkholderia cepacia CF-66 can produce cFP,which show significant inhibitory effect on the growth of Fusarium semitectum.Interestingly,our study also found that,Burkholderia seminalis R456 and its metabolites can significantly inhibit the mycelium growth of Fusarium graminearum Fg.As an antagonistic bacterium,R456 was isolated from paddy soil earlier by our laboratory.It is reasonable to speculate that cFP may be one of the active substances of antagonizing bacteria R456 to inhibit Fg.Accordingly,the following work is carried out in this study:To make clear whether the Fg R456 antagonism is partly caused by the secretion of antimicrobial substances cFP,firstly,the antagonism effect was determined by using plate confrontation method.As a result,997 of Tn5 inserted mutant strains of Fg R456 was constructed.Compared to the wild type,8 mutants(no.88,189,117,113,885,495,268 and 255)indicate improved antagonism effect(increase by more than 20%),and 5 mutants(no.216,123,85,224 and 225)show reduced antagonism effect(decreased more than 20%).Then,the supernatant filtrate of these 13 mutants was analyzed by high performance liquid chromatography(HPLC)technique.Compare to other mutants and wild type,a unique peak was observed at 20 min for mutant 88,which is consist with the peak time of synthesized cFP standard under similar experimental conditions.Further LC-MS(mass spectrometry)analysis showed relative molecular mass of 245.12,which is close to the relative molecular mass of cFP.The results of the secondary mass spectrometry showed that there were 7 debris peaks with relative molecular mass of 65.04,70.06,92.05,154.07,172.11,217.13 and 245.12,which were close to the chemical structure of cFP.Finally,the results of the plate antagonism experiment showed that both the synthetic cFP and biosynthetic cFP in strain R456 could strongly inhibit the growth of Fg mycelium,and the antagonistic effect was enhanced with the increased concentration within the range of 0.1-1000 ppm.To further explore the synthesis and regulatory mechanism of cFP in antagonistic bacteria R456,first of all,a reverse PCR amplification was applied for the above mentioned 13 mutants with antagonism effects and insertional locus was detected through blast against the genome sequence of R456.The insertional locus for mutant 88 was successfully identified for gene DctB,which is composed of Dct transfer system(Dct-A,-B,-D),and is response for the transport of four carbon dicarboxylic acid C4-dicarboxylates(dCAs).Moreover,single Dct-A,-B,or-D gene knockout mutant and complement strain was constructed,and cFP content was determined for each supernatant filtrate by using HPLC.Compared with wild type,significantly increased cFP content was observed for Dct(-A,-B or-D)mutant and no significant differences was detected for the corresponding complementary strain.It is therefore reasonable to infer that cFP synthesis of R456 is closely related to the Dct transfer system.Finally,the effect of substrate dCAs for Dct transfer system on cFP production is analyzed for R456 and its mutants by using HPLC.Significant decreased cFP production was observed for wild type strain with the presence of either tested dCAs including oxaloacetic acid,malic acid,aspartic acid and succinic acid.By contrast,there is no difference on cFP production for either of Dct-A,-B-D gene deletion mutant.Taken together,dCAs may negatively control the synthesis of cFP in strains R456.In summary,this study clearly shows that cFP is one of the main active substances of strain R456 antagonized against Fg,and its biosynthesis is negatively regulated by dCAs.更多还原