【作者】 杨柳；

【导师】 刘霞；

【作者基本信息】 武汉理工大学 ， 药学， 2022， 硕士

【摘要】 汉麻(Cannabis sativa.L)是一种历史悠久的药用植物,富集在汉麻花和苞片的部分萜酚类次生代谢产物具有重要的药用价值。其中,大麻二酚(CBD)具有治疗癫痫,镇静止痛等药效,是药用价值最高的萜酚类化合物。然而,大麻二酚(CBD)和大麻环萜酚(CBC)共用大麻萜酚酸(CBGA)前体,在代谢流关系上为竞争关系,如何通过合成生物学手段提高萜酚类化合物生物合成途径中的CBD,降低其他次要萜酚类化合物的含量是研究的重点和难点。次生代谢产物合成调控机制的阐明是合成生物学研究的基础,联合基因组和转录组挖掘萜酚类化合物合成的关键酶基因,筛选调控关键酶基因的转录因子并解析调控机制是合成调控研究的有效方法。在以前的研究中,萜酚类化合物生物合成途径中的关键酶基因已被阐明,本文结合汉麻的基因组和不同组织器官的转录组,筛选可能调控萜酚类化合物合成的APETALA2/Ethylene-Responsive Factor(AP2/ERF)转录因子和转录辅助因子组蛋白去乙酰化酶(HDAC),并对它们协同调控萜酚类化合物合成的作用机制进行研究。本研究已获得如下结果:1.筛选与萜酚类化合物合成相关的AP2/ERF转录因子结合加权基因共表达网络分析(WGCNA分析),AP2/ERF转录因子与关键酶基因的相关性分析,AP2/ERF转录因子在不同器官的特异性表达,初步筛选出3个(Cs DREB18,Cs ERF04,Cs AP2-14)可能与萜酚类化合物合成相关的AP2/ERF转录因子。对三个候选的AP2/ERF基因和关键酶基因的启动子进行双分子荧光素报告实验,发现Cs AP2-14与Cs OAC有互作,Cs DREB18与Cs OAC和Cs CBGAS都有互作,Cs AP2-14和Cs DREB18可能参与调控萜酚类化合物合成。2.鉴定汉麻的HDAC基因家族本研究鉴定了汉麻的14个HDAC基因家族成员,并对所有成员的理化性质,基因结构,染色质定位,保守结构域,可变剪切,顺式作用元件,系统进化关系等进行分析,掌握了HDAC的基本信息和家族性的功能和特点,为挖掘目标基因和功能验证打下基础。3.筛选与萜酚类化合物生物合成相关的HDAC首先分析HDAC在不同器官的基因表达模式(并用q RT-PCR验证),与关键酶基因的相关性,以及与已报道的参与次生代谢调控的HDAC同源性,进一步用组蛋白去乙酰化酶抑制剂(曲古抑菌素A,Trichostatin A,TSA)处理汉麻,对所有HDAC基因和萜酚类化合物合成通路的关键酶基因进行q RT-PCR分析,结果证明,TSA可以有效抑制HDAC基因的表达,同时影响关键酶基因的表达。综上,初步筛选出4个(Cs HDA1,Cs HDA7,Cs HDA10,Cs HDT1)可能与萜酚类化合物合成相关的HDAC。克隆候选的HDAC基因,并观察它们的亚细胞定位,发现Cs HDA1和Cs HDA10都定位在细胞核上。4.验证AP2/ERF与HDAC的互作关系通过候选的AP2/ERF转录因子与组蛋白去乙酰化酶在烟草中的荧光素酶互补(BiFC)实验,发现CsDREB18和CsHDA10有相互作用关系,它们有可能协同调控萜酚类化合物的合成。更多还原

【Abstract】 Hemp(Cannabis sativa.L)is a medicinal plant with a long history,and some terpene phenolic secondary metabolites enriched in hemp flowers and bracts have important medicinal value.Among them,CBD has the effect of treating epilepsy,analgesia and so on,CBD is the terpene phenolic compound with the highest medicinal value.However,Cannabidiol Cannabidiolic(CBD)and Cannabichromen(CBC)share the Cannabigerolic acid(CBGA)precursor,which is competitive in the metabolic flow relationship.How to improve the CBD in the biosynthetic pathway and reduce the content of other minor terpene phenolic compounds is the focus and difficulty of the research.The elucidation of the regulatory mechanism of secondary metabolite synthesis is the basis of synthetic biology research.Combining genomic and transcriptome to excavate key enzyme genes for terpene phenolic compounds synthesis,screening for transcription factors regulating key enzyme genes and resolving regulatory mechanisms are effective methods for synthetic regulation research.In previous studies,the key enzyme genes in the biosynthetic pathway of terpene phenolic compounds had been elucidated.In this paper,combining the genome of hemp and the transcriptome of different tissues and organs,we screened APETALA2/Ethylene-Responsive(AP2/ERF)transcription factors that may regulate the synthesis of terpene phenolic compounds and the transcriptional cofactor histone deacetylase(HDAC).and studied the mechanism of their synergistic regulation of terpene phenolic synthesis.The following results have been obtained in this study:1.Screening AP2/ERF transcription factors related to the synthesis of terpene phenolic compoundsCombined with weighted gene co-expression network analysis(WGCNA analysis),correlation analysis between AP2/ERF transcription factors and key enzyme genes,and the specific expression of AP2/ERF transcription factors in different organs,we screened 3 AP2/ERF transcription factors preliminarily(Cs DREB18,Cs ERF04,Cs AP2-14).They may be involved in the synthesis of terpene phenolic compounds.Binolecular fluorescein reporter experiments on the promoters of three candidate AP2 / ERF genes and key enzyme genes revealed that Cs AP2-14 interacts with Cs OAC,Cs DREB18 interacts with both Cs OAC and Cs CBGAS,and Cs AP2-14 and Cs DREB18 may be involved in the regulation of terpenoid phenolic compound synthesis.2.Identification of the HDAC gene family of hempIn this study,14 HDAC gene family members of hemp were identified.And we analyzed the physicochemical properties,gene structure,chromatin localization,conserved domains,alternative splicing,cis-acting elements,and phylogenetic relationships of all members.We master the basic information of HDAC and the functions and characteristics of this familial nature,laying a foundation for the mining of target genes and functional validation..3.Screening of HDACs related to the biosynthesis of terpene phenolic compoundsFirst,we analyzed the gene expression patterns of HDAC in different organs(verified by q RT-PCR),the correlation with key enzyme genes,and the HDAC homology reported to be involved in secondary metabolism regulation Further,we treated hemp with histone deacetylase inhibitors(trostatin A,Trichostatin A,TSA),and performed qRT-PCR analysis of all HDAC genes and terpene phenolic compound synthesis pathways,and proved that TSA could effectively inhibit the expression of HDAC genes and affect the expression of key enzyme genes.In conclusion,four HDAC(Cs HDA1,Cs HDA7,Cs HDA10,Cs HDT1)that may be related to the synthesis of terpene phenolic compounds were initially selected.We cloned the candidate HDAC genes and observed their subcellular localization,and found that both Cs HDA1 and Cs HDA10 were localized to the nucleus.4.Verify the interaction between AP2/ERF and HDACThrough luciferase complementation(BiFC)experiments of candidate AP2/ERF transcription factors and histone deacetylases in tobacco,it was found that CsDREB18 and CsHDA10 have an interacting relationship.So they may coordinately regulate the synthesis of terpene phenolic compounds.更多还原