外源乙烯調(diào)控長(zhǎng)春花萜類(lèi)吲哚生物堿合成的代謝和轉(zhuǎn)錄組分析
發(fā)布時(shí)間：2019-07-01 09:15
【摘要】：本文使用乙烯利噴施處理長(zhǎng)春花幼苗,并對(duì)其生長(zhǎng)指標(biāo)、光合指標(biāo)及生物堿含量進(jìn)行測(cè)定。同時(shí),利用HPLC-QTOF/MS和RNA-seq的技術(shù)對(duì)乙烯利處理后的長(zhǎng)春花代謝組和轉(zhuǎn)錄組進(jìn)行測(cè)定和分析,探討了乙烯利對(duì)長(zhǎng)春花內(nèi)萜類(lèi)吲哚生物堿合成的影響及分子調(diào)控。本研究取得以下成果和進(jìn)展：乙烯利抑制了長(zhǎng)春花的株高、根長(zhǎng)、根重和單株生物量,但促使莖稈直徑生長(zhǎng)增加,這種現(xiàn)象主要與乙烯的“三重反應(yīng)”有關(guān)。乙烯利處理下長(zhǎng)春花光合效率下降,所以推測(cè)乙烯利處理的長(zhǎng)春花的生物量增長(zhǎng)趨勢(shì)減緩是因?yàn)橥庠匆蚁┮种屏酥仓甑墓夂托?并且其凈光合速率的下降由非氣孔因素造成的。外源乙烯對(duì)長(zhǎng)春花光合色素含量也有調(diào)控作用,促使長(zhǎng)春花葉綠素含量降低,類(lèi)胡蘿卜素含量升高。也從側(cè)面說(shuō)明葉綠素含量降低也是導(dǎo)致長(zhǎng)春花凈光合速率下降的原因之一,進(jìn)而使其單株生物量降低。對(duì)于長(zhǎng)春花的四種萜類(lèi)吲哚生物堿,乙烯利促進(jìn)異胡豆苷和長(zhǎng)春堿的合成,長(zhǎng)春堿合成的前體長(zhǎng)春質(zhì)堿和文多靈含量的變化不同,長(zhǎng)春質(zhì)堿含量上升,文多靈含量略有下降,但其終產(chǎn)物長(zhǎng)春堿含量是上升的。乙烯利處理使初生代謝物UDP-L-鼠李糖和L-苯丙氨酸含量有所下降,卻促進(jìn)次生代謝相關(guān)的初生代謝物色氨酸的合成。色氨酸是莽草酸途徑的初始代謝物,直接影響了下游異胡豆苷的合成。參與到2-C-甲基-D-赤蘚糖醇-4-磷酸鹽(MEP)代謝途徑的1-羥基-2甲基-2(E)丁烯基-4磷酸氫鹽也被乙烯利調(diào)控上升,而MEP途徑也是莽草酸途徑的上游代謝途徑。乙烯利對(duì)長(zhǎng)春花次生代謝的調(diào)節(jié)作用十分明顯,異胡豆苷(Strictosidine)、蛇根堿(Serpentine)、長(zhǎng)春質(zhì)堿(Catharanthine)及長(zhǎng)春堿(Vinblastine)等萜類(lèi)吲哚生物堿合成途徑中的代謝產(chǎn)物含量均呈上升的趨勢(shì)。代謝流由初生代謝向次生代謝轉(zhuǎn)變的趨勢(shì),揭示了代謝通路之間的相互作用。長(zhǎng)春花初生代謝途徑和次生代謝途徑中合成酶相關(guān)基因在乙烯利處理后上調(diào)表達(dá),這也解釋了乙烯利促進(jìn)一部分生物堿合成的中間產(chǎn)物和終產(chǎn)物的合成。轉(zhuǎn)錄組數(shù)據(jù)分析的結(jié)果表明以代謝過(guò)程來(lái)講,共30061個(gè)基因(轉(zhuǎn)錄本)參與到了401個(gè)KEGG代謝途徑中,并且篩選了195個(gè)與萜類(lèi)吲哚生物堿合成相關(guān)的基因。這195個(gè)Unigenes能夠歸屬到4個(gè)主要類(lèi)別,包括萜類(lèi)吲哚生物堿合成途徑相關(guān)基因,轉(zhuǎn)錄因子基因,ABC轉(zhuǎn)運(yùn)蛋白和MATE轉(zhuǎn)運(yùn)蛋白基因,基本都是參與次生代謝生物合成途徑的基因,特別是類(lèi)單萜吲哚生物堿合成途徑相關(guān)基因。進(jìn)一步分析這195個(gè)與萜類(lèi)吲哚生物堿合成相關(guān)的基因,結(jié)果表明195個(gè)基因中的32個(gè)在乙烯利處理后上調(diào)表達(dá),35個(gè)基因在乙烯利處理后是下調(diào)的；32個(gè)上調(diào)表達(dá)基因主要功能是金屬離子結(jié)合活性和裂解酶活性,35個(gè)下調(diào)表達(dá)基因的分子功能集中在甲基化酯酶活性。這種乙烯利處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現(xiàn)象可能與TIAs合成相關(guān)基因的高表達(dá)有關(guān),但還需進(jìn)一步證實(shí)。通過(guò)主成分分析得到TIAs合成途徑中催化酶基因、轉(zhuǎn)錄因子基因和生物堿合成量的相關(guān)關(guān)系,分析表明CrWRKY1基因的轉(zhuǎn)錄水平與基因ZCT1,ZCT2和ORCA3存在明顯相關(guān)性,可以推斷WRKY基因除了具有廣泛地反饋調(diào)控外,還可以利用其他信號(hào)通路的轉(zhuǎn)錄因子來(lái)調(diào)節(jié)其轉(zhuǎn)錄活性,MAPK的表達(dá)量與其他轉(zhuǎn)錄因子明顯相關(guān),并且與其他調(diào)控因子例如激活子、抑制子等共同對(duì)萜類(lèi)吲哚生物堿上游合成途徑的關(guān)鍵酶基因有調(diào)控作用,進(jìn)而調(diào)控萜類(lèi)吲哚生物堿合成。催化酶基因、生物堿與轉(zhuǎn)錄因子基因三者之間相關(guān)性分析表明轉(zhuǎn)錄因子ORCA3和ZCT1、催化酶基因T16H和蛇根堿(ser)之間存在明顯的相關(guān)性。ORCA3與ZCT1之間呈明顯的負(fù)相關(guān),可以推斷轉(zhuǎn)錄因子的競(jìng)爭(zhēng)性作用協(xié)調(diào)生物堿合成相關(guān)基因的表達(dá)。蛇根堿和長(zhǎng)春堿的含量與次生代謝下游的催化酶基因和轉(zhuǎn)錄因子有明顯相關(guān)性。萜類(lèi)吲哚生物堿合成途徑上的基因表達(dá)與轉(zhuǎn)錄因子的基因表達(dá)緊密相關(guān),使代謝通路可以被外源乙烯激活,促進(jìn)生物堿的積累。
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q946;Q943.2
[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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：Q946;Q943.2
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