外源乙烯调控长春花萜类吲哚生物碱合成的代谢和转录组分析

发布时间：2019-07-01 09:15

【摘要】：本文使用乙烯利喷施处理长春花幼苗,并对其生长指标、光合指标及生物碱含量进行测定。同时,利用HPLC-QTOF/MS和RNA-seq的技术对乙烯利处理后的长春花代谢组和转录组进行测定和分析,探讨了乙烯利对长春花内萜类吲哚生物碱合成的影响及分子调控。本研究取得以下成果和进展：乙烯利抑制了长春花的株高、根长、根重和单株生物量,但促使茎秆直径生长增加,这种现象主要与乙烯的“三重反应”有关。乙烯利处理下长春花光合效率下降,所以推测乙烯利处理的长春花的生物量增长趋势减缓是因为外源乙烯抑制了植株的光和效率,并且其净光合速率的下降由非气孔因素造成的。外源乙烯对长春花光合色素含量也有调控作用,促使长春花叶绿素含量降低,类胡萝卜素含量升高。也从侧面说明叶绿素含量降低也是导致长春花净光合速率下降的原因之一,进而使其单株生物量降低。对于长春花的四种萜类吲哚生物碱,乙烯利促进异胡豆苷和长春碱的合成,长春碱合成的前体长春质碱和文多灵含量的变化不同,长春质碱含量上升,文多灵含量略有下降,但其终产物长春碱含量是上升的。乙烯利处理使初生代谢物UDP-L-鼠李糖和L-苯丙氨酸含量有所下降,却促进次生代谢相关的初生代谢物色氨酸的合成。色氨酸是莽草酸途径的初始代谢物,直接影响了下游异胡豆苷的合成。参与到2-C-甲基-D-赤藓糖醇-4-磷酸盐(MEP)代谢途径的1-羟基-2甲基-2(E)丁烯基-4磷酸氢盐也被乙烯利调控上升,而MEP途径也是莽草酸途径的上游代谢途径。乙烯利对长春花次生代谢的调节作用十分明显,异胡豆苷(Strictosidine)、蛇根碱(Serpentine)、长春质碱(Catharanthine)及长春碱(Vinblastine)等萜类吲哚生物碱合成途径中的代谢产物含量均呈上升的趋势。代谢流由初生代谢向次生代谢转变的趋势,揭示了代谢通路之间的相互作用。长春花初生代谢途径和次生代谢途径中合成酶相关基因在乙烯利处理后上调表达,这也解释了乙烯利促进一部分生物碱合成的中间产物和终产物的合成。转录组数据分析的结果表明以代谢过程来讲,共30061个基因(转录本)参与到了401个KEGG代谢途径中,并且筛选了195个与萜类吲哚生物碱合成相关的基因。这195个Unigenes能够归属到4个主要类别,包括萜类吲哚生物碱合成途径相关基因,转录因子基因,ABC转运蛋白和MATE转运蛋白基因,基本都是参与次生代谢生物合成途径的基因,特别是类单萜吲哚生物碱合成途径相关基因。进一步分析这195个与萜类吲哚生物碱合成相关的基因,结果表明195个基因中的32个在乙烯利处理后上调表达,35个基因在乙烯利处理后是下调的；32个上调表达基因主要功能是金属离子结合活性和裂解酶活性,35个下调表达基因的分子功能集中在甲基化酯酶活性。这种乙烯利处理后甲基化酶的活性大幅降低但裂解酶的活性大幅增加的现象可能与TIAs合成相关基因的高表达有关,但还需进一步证实。通过主成分分析得到TIAs合成途径中催化酶基因、转录因子基因和生物碱合成量的相关关系,分析表明CrWRKY1基因的转录水平与基因ZCT1,ZCT2和ORCA3存在明显相关性,可以推断WRKY基因除了具有广泛地反馈调控外,还可以利用其他信号通路的转录因子来调节其转录活性,MAPK的表达量与其他转录因子明显相关,并且与其他调控因子例如激活子、抑制子等共同对萜类吲哚生物碱上游合成途径的关键酶基因有调控作用,进而调控萜类吲哚生物碱合成。催化酶基因、生物碱与转录因子基因三者之间相关性分析表明转录因子ORCA3和ZCT1、催化酶基因T16H和蛇根碱(ser)之间存在明显的相关性。ORCA3与ZCT1之间呈明显的负相关,可以推断转录因子的竞争性作用协调生物碱合成相关基因的表达。蛇根碱和长春碱的含量与次生代谢下游的催化酶基因和转录因子有明显相关性。萜类吲哚生物碱合成途径上的基因表达与转录因子的基因表达紧密相关,使代谢通路可以被外源乙烯激活,促进生物碱的积累。
[Abstract]:In this paper, the growth index, the photosynthetic index and the alkaloid content of the Catharanthus roseus seedlings were treated with ethrel. At the same time, the effects of ethrel on the synthesis of the alkaloids in the vinblastine and the regulation of the molecular control were discussed by means of HPLC-QTOF/ MS and RNA-seq technique. The following results and progress have been achieved in this study: the plant height, the root length, the root weight and the single plant biomass of the Catharanthus roseus are inhibited, but the diameter of the stem is increased, and the phenomenon is mainly related to the "triple reaction" of ethylene. The decline of the photosynthetic efficiency of Catharanthus roseus under the treatment of ethrel, therefore, it was suggested that the growth of the biomass of the Catharanthus roseus was slower because the exogenous ethylene inhibited the light and the efficiency of the plant, and the decrease of the net photosynthetic rate was caused by non-stomatal factors. The effects of exogenous ethylene on the content of the photosynthetic pigment of the catharanthus roseus were also controlled, and the content of chlorophyll in the catharanthus roseus was reduced and the content of the carotenoid increased. The decrease of the content of chlorophyll from the side is also one of the reasons leading to the decrease of the net photosynthetic rate of the catharanthus roseus. The contents of vinblastine and vinblastine in vinblastine in the synthesis of vinblastine and vinblastine are different from that of vinblastine and vinblastine. But the vinblastine content of the end product of the final product is increased. The content of the primary metabolite UDP-L-rhamnose and L-phenylalanine decreased, but the synthesis of the primary metabolite tryptophan associated with secondary metabolism was promoted. Tryptophan is the initial metabolite of shikimic acid pathway, which directly affects the synthesis of downstream isohubarb. The 1-hydroxy-2-methyl-2 (E) butenyl-4-phosphate salt of 2-C-methyl-D-erythritol-4-phosphate (MEP) metabolic pathway is also subject to an increase in the regulation of ethylene, and the MEP approach is also an upstream metabolic pathway of the shikimic acid pathway. The effect of ethrel on the secondary metabolism of the catharanthus roseus is very obvious, and the content of the metabolites in the synthetic pathway of the alkaloids such as the rictosidine, serpentine, catharanthine and vinblastine is on the rise. The tendency of metabolic flow to transition from primary to secondary metabolism reveals the interaction between metabolic pathways. The primary metabolic pathway of the vinblastine and the associated gene of the synthetase in the secondary metabolic pathway are up-regulated after the treatment with ethylene, which also explains the synthesis of the intermediate and end products of the synthesis of a part of the alkaloid from the ethrel. The results of the data analysis of the transcriptome showed that, in the process of metabolism, a total of 30061 genes (transcripts) were involved in the 401 KEGG metabolic pathways, and 195 of the genes associated with the synthesis of the total alkaloids of the same class were screened. The 195 Universies can be attributed to the four main categories, including the genes related to the synthetic pathway of the total alkaloids of the class 1, the transcription factor gene, the ABC transporter and the MATE transporter gene, and are basically the genes involved in the secondary metabolism biosynthetic pathway, In particular to a gene which is related to that synthetic route of the alkaloid of the monotype monochamus. The results showed that 32 of the 195 genes were up-regulated and 35 genes were down-regulated after they were treated with ethylene. The main functions of the 32 up-regulated genes are metal ion binding activity and lytic enzyme activity, and the molecular functions of 35 down-regulated expression genes are concentrated in the methylation esterase activity. The activity of methylase after the ethylene-extraction treatment is greatly reduced, but the phenomenon that the activity of the lysing enzyme is greatly increased may be related to the high expression of the TIAs synthesis-related gene, but further confirmation is required. The correlation between the catalytic enzyme gene, the transcription factor gene and the total alkaloid synthesis in the TIAs synthesis pathway is obtained by the principal component analysis, and the analysis shows that the transcription level of the CrWRKY1 gene is obviously related to the genes ZCT1, ZCT2 and ORCA3, and the WRKY gene can be inferred that the WRKY gene has wide feedback regulation and control, the transcription activity of other signal paths can also be adjusted, the expression amount of the MAPK is obviously related to other transcription factors, So as to control the synthesis of the total alkaloids of the monotype of the Congo. The correlation between the catalytic enzyme gene, the alkaloid and the transcription factor gene indicates that the transcription factors ORCA3 and ZCT1, the catalytic enzyme gene T16H and the snake root base (ser) have a clear correlation. There is a significant negative correlation between ORCA3 and ZCT1, and it can be deduced that the competitive action of transcription factor can coordinate the expression of the related genes in the synthesis of alkaloids. The contents of the serpent and the vinblastine have a significant correlation with the catalytic enzyme gene and the transcription factor downstream of the secondary metabolism. The expression of the gene on the synthetic pathway of the total alkaloid of the Astragalus is closely related to the gene expression of the transcription factor, so that the metabolic pathway can be activated by the exogenous ethylene to promote the accumulation of the alkaloid.
【学位授予单位】：东北林业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q946;Q943.2

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