【作者】 曾桂萍；

【导师】 赵致；

【作者基本信息】 贵州大学 ， 生理学， 2019， 博士

【摘要】 中国传统原药茯苓是多孔菌科真菌Wolfiporia cocos的干燥菌核,是一种药食两用的大宗药材,药用历史长且具有广泛而重要的药用价值。现代研究表明,茯苓的活性成分三萜具有多种药理作用。茯苓的研究也多集中在药用成分及药理功效等方面,转录组及分子水平研究较少。提高茯苓中三萜类化合物的生物合成和积累,选育高产三萜的茯苓菌种是目前研究的热点。本研究通过有性产孢条件的优化、单孢菌株的分离筛选等传统方法,结合转录组学、代谢组学等现代方法综合研究,揭示茯苓三萜类化合物生物合成的关键基因及其调控因子,分析其与三萜相关代谢物的关联性,进而获得茯苓三萜类化合物生物合成的调控基因。为生产上提高发酵生产茯苓三萜的产量和高产三萜茯苓的菌株育种奠定科学基础。本研究获得如下主要结果:1.茯苓有性产孢的条件优化为了构建茯苓近等基因系,获得遗传基础一致,总三萜类化合物含量不同的差异菌株,本研究优化了实验室培养茯苓的有性产孢条件。分别对培养基、培养容器、培养基初始p H值、温度和光照等条件进行单因素实验。获得最优产孢条件是:在装有p H 6.0～7.0的茯苓马铃薯葡萄糖琼脂(PDA)培养基A的培养瓶中,经过25～28℃7 d暗培养,38 d光暗交替培养后,产孢量可达(8.12±0.11)×10~9个/瓶。2.筛选出茯苓总三萜含量差异显著的两个单孢菌株为了避免研究材料遗传背景和不同生长发育阶段的差异对研究结果的影响,本研究通过单孢分离法培养,从同一茯苓菌株5.78的有性繁殖子代236个单孢菌株中,筛选出了总三萜产量差异显著的高产(H)和低产(L)两个单孢菌株。3.茯苓菌丝的转录组分析对茯苓两个菌株(H&L),三个培养时间段(培养菌丝17 d、34 d和51 d),3次生物学重复共18个样本进行转录组测序。转录本组装基因总数为16879个,其中12260个(72.63%)被NR等数据库注释。可编码蛋白基因中有96.51%是已知蛋白,氨基酸数大于300的蛋白质占42.96%。SSR显著偏好GC重复,以三核苷酸重复单元(CCG/CGG)n为主。两组菌株组间差异表达基因中与三萜类合成通路相关的基因有FDPS、erg11、erg26、erg6和SQLE,在三萜类合成通路之外的基因Pm20d2和nor A等,推测它们与三萜类生物合成有关。转录组的短时间序列表达(STEM)分析结果获得了10个三萜类化合物合成相关通路上的核心基因(HMGCR、COQ2、ERG2、ACAT、TAT、erg11、erg26、SQLE、CAO2和FACE1)以及7个相关的核心基因(Pm20d2、nor A、染色质重塑ATP酶基因、adh、ftm P450-1、未知蛋白unigene0011374和unigene0001029)。核心基因大多属于膜蛋白或是甾醇和细胞膜稳态的关键组成部分的相关基因。维持茯苓三萜类化合物高积累可能需要有稳定的膜结构做保证,高积累三萜的能力可能与固醇的合成能力有关。转录组的权重基因共表达网络分析(WGCNA)结果得到与三萜类化合物的合成和积累相关的通路上有ACAT1-b、hgs A、mvd1、SQLE、erg11、TAT、erg26和2个erg6共9个核心基因,通路外有Pm20d2和nor A等多个调控因子和蛋白基因。这些基因的表达说明茯苓的三萜类化合物的合成与积累与甾醇的生物合成有着紧密的联系。三萜类化合物合成相关通路上的SQLE、TAT、erg26和erg11,以及通路外的Pm20d2和nor A这6个基因是在以上两种分析方法都表现出极高的相关性和连通性的关键基因。4.茯苓菌丝代谢组分析对茯苓两个菌株(H&L),三个培养时间段(培养菌丝17 d、34 d和51 d),6次生物学重复共36个样本进行代谢组分析。LC-MS分析共检查到正负离子8219种,其中已知物质75914种,占比92.36%。差异代谢物KEGG富集结果证实了转录组的分析结果,三萜类化合物的生物合成与积累,是与固醇的合成、维持膜结构、膜转运功能是密切联系的。代谢物的STEM分析结果表明茯苓的三萜类化合物和多糖的合成和积累存在着此消彼长的情况。差异基因与差异代谢物的关联分析发现FDPS、CAO2、erg11和Pm20d2是经代谢组分析证实的调控基因。5.调控基因的蛋白质生物学信息分析针对Pm20d2和erg11两个呈紧密负相关的调控基因进行蛋白质生物信息分析,结果表明它们都不是分泌蛋白,都有一个编码蛋白具有跨膜域,二级结构大多是以无规则卷曲为主,三级结构差别迥异,是结构和功能不同的编码蛋白,都是定位在细胞质膜上执行功能的蛋白。Pm20d2系统发育关系分析证实了转录组基因序列同源性分析结果,erg11却是单源发生,与其他菌种有一定遗传距离,具有独特性。6.三萜类化合物合成与积累调控机制分析筛选到的关键基因的表达情况说明,三萜类化合物生物合成的基因调控可能发生在从转录到翻译后加工的多个水平上。三萜类化合物生物合成不仅受到上下游基因的调控,还受到支路和通路外基因的调控。综上所述,本研究通过转录组和代谢组分析,筛选到1个注释在三萜类化合物合成通路上的调控基因erg11(unigene0015621)和与它紧密负相关的1个通路外的调控基因Pm20d2,与三萜类化合物合成具有共同前体的其他支路的基因表达同时间接地调控三萜类化合物的合成和积累。维持茯苓三萜类化合物高积累可能需要有稳定的膜结构做保证;三萜类化合物和多糖的合成和积累存在此消彼长的情况。本研究结果为生物工程生产茯苓三萜奠定了理论基础,也为其他材料生产三萜类化合物提供理论参考。研究结果对提高三萜产量有重要意义,对高产三萜材料的选育也有重要意义。更多还原

【Abstract】 Wolfiporia cocos(Schwein.)Ryvarden&Gilb,belongs to the genus Wolfiporia of the familyPolyporaceae.It is a traditional Chinese medicine(TCM)that is always applied both medicinal and edible and has long medicinal history,which is the dried sclerotia of Polyporaceae fungal W.cocos.Modernresearch have shown that triterpenoid,the active ingredient of W.cocos,have many pharmacological effects.Researches of W.cocos mainly focus on the aspects of medicinal ingredients and pharmacological effects,while few researches on transcriptome and molecular level.It is a hot research topic to improve thebiosynthesis and accumulation of triterpenoid in W.cocos and to select the strains with high triterpenoid production.In present study,through the traditional methods such as optimization of sexual sporulation conditions,the isolation and screening of single spore strains,combined with transcription,metabonomics and othermodern methods,a comprehensive study revealed key genes of W.cocos triterpenoid biosynthesis and its regulation factors,and analyze their relation with triterpenoid,then,the regulatory genes of W.cocostriterpenoid biosynthesis were obtained.It lays a scientific foundation for improving the yield oftriterpenoid and the breeding of strains with high yield of triterpenoid in the future.The main results are as follows:1.Condition optimization of W.cocos sexual sporulationIn order to construct a near isogenic line,obtain different strains with consistent genetic basis anddifferent total content of triterpenoid,this study has optimized the conditions for sexual spore production of W.cocos in laboratory culture.Single factor experiments were carried out on medium,culture container,intial medium p H value,temperature and light conditions.The optimal condition of spore production of W.cocos is(8.12±0.11)×10~9/bottle in the culture bottle of PDA medium A with p H 6.0～7.0,after dark culture of 25～28℃for 7 days and alternating culture of light and dark for 38 days.2.Two single spore strains with significant difference in total triterpenoid content of W.cocos were screened out.In order to avoid material differences in genetic background and different growth stages to influence on the results of this study,two monosporal strains,high yield(H)and low yield(L),were screened out from the 236 sexual reproductive progenies of the same W.cocos 5.78.3.Transcriptome analysis of W.cocos myceliaTwo strains of W.cocos(H&L),three times of culture(cultured mycelia for 17 days,34 days,and 51days),3 times of biological repetition,a total of 16879 genes were assembled from 18 transcripts,of which12260(72.63%)were annotated by NR database.96.51%of the encoded protein genes are known proteins,and 42.96%are proteins with amino acid number greater than 300.SSR shows a significant preference for GC repeat,dominated by trinucleotide repeat unit(CCG/CGG)n.Among the differentially expressed genes between the two groups,FDPS,erg11,erg26,erg6 and SQLE were related to the triterpene synthesispathway,while Pm20d2 and nor A were outside the triterpene synthesis pathway,which were speculated to be related to triterpenoid biosynthesis.In this study,10 core genes(HMGCR,COQ2,ERG2,ACAT,TAT,erg11,erg26,SQLE,CAO2 and FACE1)and 7 related core gene(Pm20d2,nor A,chromatin remodeling ATPase genes,adh,ftmp450-1,unknown proteins Unigene0011374 and unigene0001029)were obtained by transcriptional STEM analysis.Most of the core genes belong to the gene of membrane proteins or sterols and the key components of cell membrane homeostasis.Maintaining high triterpenoid accumulation in W.cocos may also require a stable membrane structure,so the accumulation ability may be related to the high synthesis ability of sterols.According to the results of WGCNA analysis in this study,there were 9 core genes related to thesynthesis and accumulation of triterpenoid,such as ACAT1-b,hgs A,mvd1,SQLE,erg11,TAT,erg26 and two erg6,and outside the pathway there were multiple regulatory factors and protein genes such as Pm20d2and nor A.Their expressions indicated that the synthesis and accumulation of triterpenoid of W.cocos were closely related to the biosynthesis of sterol.The genes of SQLE,TAT,erg26 and erg11 in the relatedpathways of triterpenoid synthesis,as well as Pm20d2 and nor A outside the pathways,showed extremely high correlation and connectivity in both analytical methods.4.Analysis of metabolism of mycelia in W.cocosA total of 8219 positive and negative ions were detected by LC-MS analysis of a total of 36 samples from two strains(H&L),three culture times(cultured mycelia for 17 days,34 days,and 51 days)and 6times of biological repetition,among which 75914 were known substances,accounting for 92.36%.The enrichment results of differential metabolite KEGG confirmed the results of transcriptome analysis,the biosynthesis and accumulation of triterpenoid were closely related to the synthesis of sterols,maintenance of membrane structure and membrane transport function.The results of STEM analysis of metabolitesindicated that the synthesis and accumulation of triterpenoid and polysaccharides in W.cocos were trading off and taking turns.Correlation analysis of differentially expressed genes and differentially expressedmetabolites showed that FDPS,CAO2,erg11(unigene0015621)and Pm20d2 were the regulatory genes confirmed by metabolic analysis.5.Biological information analysis of regulatory genesThe results of biological information analysis for two genes of Pm20d2 and erg11 showed that all of them are not secreted proteins,but have one coding protein with transmembrane domain.Their secondary structure are mainly random coil,and their tertiary structure are very different,which are the coding protein with different structure and function,and they all are that perform functions on the membrane.Phylogenetic relationship analysis of Pm20d2 confirmed the homology analysis results of transcriptomegene sequence;erg11 was a single source,with a certain genetic distance from other strains and uniqueness.6.Analysis on the regulation mechanism of biosynthesis and accumulation of triterpenoidThe expression of key genes indicated that gene regulation of triterpenoid synthesis may occur atvarious levels from transcription to post-translation processing.The gene expression of other branches with common precursors indirectly regulates the synthesis and accumulation of triterpenoid.Regulatory factors outside the pathway of triterpenoid synthesis also play an important role in the biosynthesis of triterpenoid.In conclusion,through the transcriptome and metabolomics analysis,this study found that a regulatory gene erg11(unigene0015621)annotated in the triterpenoid synthesis pathway and a regulatory genePm20d2 out of the pathway,they were closely negatively correlated with.The synthesis and accumulation of triterpenoid are closely related to the biosynthesis of sterols.The gene expression of other branches with common precursors indirectly regulates the synthesis and accumulation of triterpenoid.Maintaining thehigh triterpenoid accumulation of W.cocos may require a stable membrane structure.The synthesis and accumulation of triterpenoid and polysaccharides are trading off and taking turns.The results of this study lay a theoretical foundation for the production of triterpenoid with W.cocos in biological engineering and also provide a theoretical reference for the production of triterpenoid with other materials.The results are of great significance to improve the yield of triterpenoid and breed of triterpenoid strain with high yield.更多还原