ISWC OpenIR  > 水保所2018--2019届毕业生论文
丹参转录因子bHLH7与MYB39互作调控酚酸类及丹参酮类物质代谢分子机制研究
邢丙聪
Subtype博士
Thesis Advisor梁宗锁
2019-05-27
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Name理学博士
Degree Discipline生态学
Keyword丹参 次生代谢 Bhlh Myb 协同调控
Abstract

丹参中次生代谢物酚酸类和丹参酮类成分的含量是评价丹参药材品质的重要指标,这两类物质的生物合成受到环境因子和遗传因素的双重影响。转录因子不但可以直接调控遗传基因的表达,还介导了环境因子对植物次生代谢物质合成的调控。因此,揭示转录因子在丹参次生代谢中的调控机制是丹参代谢工程研究的前提,也可以为阐明丹参药材品质形成机制奠定基础。

本论文以毛状根为材料,对丹参5个bHLH家族转录因子调控酚酸类和丹参酮类物质的分子机制进行了研究,并阐述了SmbHLH7与SmMYB39两个转录因子对酚酸类物质代谢的协同调控机制。具体结果如下:

  1. 我们从丹参转录组数据库中筛选到5个完整的bHLH家族转录因子,它们都包含了典型的HLH结构域,分别命名为:SmbHLH3、SmbHLH7、SmbHLH148、SmbHLH10和SmbHLH130。这几个转录因子基因在丹参植株六个不同部位(花、叶、茎、根皮、中柱、须根)均有表达,但是表达模式存在差异;同时,这几个转录因子基因不同程度的响应了MeJA、ABA和GA的诱导。亚细胞定位分析发现5个转录因子除SmbHLH10在细胞质中也有分布外,都位于细胞核中。

  2. 分别获得了SmbHLH3SmbHLH7SmbHLH148SmbHLH10SmbHLH130的过表达毛状根,检测发现SmbHLH3负调控了三种酚酸(咖啡酸、迷迭香酸、丹酚酸B)和四种丹参酮(隐丹参酮、二氢丹参酮Ⅰ、丹参酮Ⅰ、丹参酮ⅠⅠA)成分的代谢;SmbHLH7、SmbHLH148和SmbHLH130对酚酸类和丹参酮类物质的积累都是促进的作用;SmbHLH10对酚酸类成分代谢无显著调控作用,但促进了丹参酮类成分的生物合成。转基因毛状根中酚酸类和丹参酮类物质生物合成途径上的关键酶基因表达相对于野生型也发生了显著的变化。利用酵母单杂交实验发现SmbHLH3可以与酚酸类物质代谢途径上关键基因TATHPPR及丹参酮类物质代谢途径上关键基因KSL1CYP76AH1启动子区的G-box元件结合;SmbHLH7、SmbHLH148和SmbHLH130都可以结合到酚酸类物质代谢途径上C4H1TATHPPRCYP98A14和丹参酮类物质途径上DXS2CPS1CYP76AH1的启动子区;此外,SmbHLH148还可以结合到PAL1启动子上的G-box,SmbHLH7和SmbHLH130还可以结合到KSL1基因启动子区,但SmbHLH130与KSL1CYP76AH1基因上的G-box结合较弱;SmbHLH10可与CPS1CPS5DXS2的启动子序列互作。

  3. 前期我们研究发现了一个负调控酚酸类物质代谢的R2R3 MYB转录因子:SmMYB39,鉴于植物中bHLH转录因子通常会与MYB蛋白互作形成复合体调控次生代谢进行,我们利用酵母双杂交的方式对5个SmbHLH进行了筛选。结果显示, SmbHLH7和SmbHLH130都可以与SmMYB39互作,但是包含pGBKT7-SmbHLH130与pGADT7-SmMYB39的酵母菌株生长较弱。对SmbHLH7和SmMYB39蛋白序列分析发现其互作区域可能位于各自N端,蛋白截短实验验证了这一猜测。

  4. 分别获得了SmMYB39过表达和RNAi干扰的转基因毛状根,发现SmMYB39还负调控了丹参酮类成分的积累。SmbHLH7的RNAi沉默毛状根中酚酸类和丹参酮类物质的含量相对于对照都显著减少。分析发现SmMYB39和SmbHLH7调控酚酸类和丹参酮类物质代谢的基因具有重叠。将SmbHLH7SmMYB39共表达发现SmMYB39的表达量可以提高到对照的几百倍,而SmbHLH7的表达只提高了近10倍。在过表达SmbHLH7的同时将SmMYB39沉默后酚酸类和丹参酮物质的代谢得到促进,而且促进效果要高于单独过表达SmbHLH7或单独沉默SmMYB39SmMYB39的过表达会抑制SmbHLH7的转录,沉默SmMYB39SmbHLH7的表达得到提高;而过表达SmbHLH7会促进SmMYB39的转录,沉默SmbHLH7SmMYB39的转录受到抑制。说明两个转录因子之间除在蛋白层面有相互调节之外,在转录层面也可以直接互相调控。

    本文通过对丹参转录组数据分析,筛选出5个参与酚酸类或丹参酮类物质生物合成调控的SmbHLH基因,利用毛状根转基因技术对其功能进行了解析,并对这几个转录因子的靶基因进行了鉴定。还发现SmbHLH7与负调控因子SmMYB39对丹参活性成分代谢协同调控机制有三个层面:SmMYB39与SmbHLH7竞争性的结合酚酸或丹参酮代谢途径关键基因;负调控因子SmMYB39直接抑制SmbHLH7转录;SmMYB39与其他正调控的MYB竞争性结合SmbHLH7,使得正调控MBW复合体无法形成。

Other Abstract

Contents of phenolic acids and tanshinones in Salvia miltiorrhiza Bunge are the key quality indicator to evaluate Chinese herb medicine, Danshen. It is very important to illuminate the regulation mechanism of phenolic acids and tanshinones biosynthesis which could help us got high quality Danshen. Biosynthesis of phenolic acids and tanshinones are affected by both environment and genetic. Transcription factors (TFs) could regulate genes’ expression directly as well as take part in the regulation of environment factors on plant secondary metabolism. Reveal the regulation mechanism of TFs on S. miltiorrhiza secondary metabolism is the premise of S. miltiorrhiza metabolism engineering research and can also lay a foundation for elucidation of the quality formation mechanism of S. miltiorrhiza.

In this study, the molecular mechanism of 5 bHLH family TFs of S. miltiorrhiza on regulating phenolic acids and tanshinones was studied, and the synergistic regulation mechanism of SmbHLH7 and SmMYB39 on phenolic acids was described. The specific results are as follows:

1. Five complete TFs gene of the bHLH family were screened from the S. miltiorrhiza transcriptome database, which were named as SmbHLH3, SmbHLH7, SmbHLH148, SmbHLH10 and SmbHLH130, respectively. Typical HLH domain were found in these five TFs by comparative analysis with other databases. All these five bHLHs present in the six parts (stem, leaf, flower, root epidermis, xylem and fibrous root) of two years old flowering S. miltiorrhiza, but with different expression patterns. The five bHLHs expression were also being induced by MeJA, ABA and GA. Results of subcellular localization showed that GFP fluorescence of these proteins all present in nucleus, and fluorescence of SmbHLH10-GFP also present in other places in the cell with lower luminance.

2. The gene overexpression hairy roots of five SmbHLHs were obtained, respectively. We found that overexpression of SmbHLH3 inhibited the accumulation of both tanshinones and phenolic acids in S. miltiorrhiza hairy roots. Phenolic acids and tanshinones biosynthesis were induced in transgenic hairy roots of SmbHLH7, SmbHLH148 and SmbHLH130, respectively. Overexpression of SmbHLH10 improved tanshinones accumulation while showed non-significant effect on phenolic acids biosynthesis. The main enzyme genes’ expression of phenolic acids and tanshinones biosynthetic pathways were changed significantly in transgenic hairy roots when compared to WT. Results of yeast one-hybrid assay showed that SmbHLH3 could binds the predicted G-box motifs within the promoters of TAT and HPPR, while SmbHLH7, SmbHLH148, SmbHLH130 could binds the promoters of C4H1, TAT, HPPR, CYP98A14. Moreover, SmbHLH148 could also binds the G-box motifs within the promoters of PAL1. For tanshinones biosynthesis pathway, SmbHLH3 could interact with promoters of KSL1 and CYP76AH1, SmbHLH7, SmbHLH148 and SmbHLH130 could interact with promoters of DXS2, CPS1, CYP76AH1. SmbHLH7 and SmbHLH130 could also interact with G-box within KSL1 promoter. The interactions of SmbHLH130 with KSL1 or CYP76AH1 were weaker than others. SmbHLH10 could binds the predicted G-box motifs within the promoters of DXS2, CPS1 and CPS5.

3. Previously we found that SmMYB39 was a R2R3MYB which regulated phenolic acids biosynthesis negatively. Considering that bHLH transcription factors generally interact with MYB protein to form a complex to regulate secondary metabolism, we screened these 5 SmbHLHs whether it could interact with SmMYB39 by yeast two-hybrid assay. The results showed that SmbHLH7 and SmbHLH130 could both interact with SmMYB39. The interaction between SmbHLH130 and SmMYB39 was weakly. The sequence analysis of SmbHLH7 and SmMYB39 proteins showed that the interaction region of SmbHLH7 and SmMYB39 protein may located at the N terminal of each other, which was confirmed by yeast two-hybrid experiment after the protein was truncated.

4. We got the overexpression and RNAi silencing hairy roots of SmMYB39. Found that SmMYB39 suppressed tanshinones biosynthesis as well as phenolic acids. We also got RNAi silencing hairy roots of SmbHLH7, in which phenolic acids and tanshinones contents were decreased when compared with WT. Analyzed of genes expression of phenolic acids and tanshinones metabolic pathways in transgenic hairy roots revealed that SmMYB39 and SmbHLH7 could regulate the same genes. The co-overexpression of SmbHLH7 and SmMYB39 showed that the expression level of SmMYB39 was increased to several hundred times of the control group, while the expression level of SmbHLH7 was only increased by less than 10 times. The biological synthesis of phenolic acids was inhibited, while the content of tanshinone was increased. When SmbHLH7 was overexpressed and SmMYB39 was silenced at the same time, the accumulation of phenolic acids and tanshinones were also promoted, and the promotion ratio compared to WT were higher than that of the hairy root of SmbHLH7 overexpression or SmMYB39 RNAi alone. The transcription of SmbHLH7 was inhibited in the overexpressed hairy roots of SmMYB39, while it was increased after the silencing of SmMYB39. Conversely, overexpression of SmbHLH7 promotes transcription of SmMYB39, which was inhibited by silencing SmbHLH7. These results indicated that in addition to the interaction between SmMYB39 and SmbHLH7 at proteins level, they could co-adjust at transcription level.

In the present study, we got five bHLHs from S. miltiorrhiza transcriptome database which could regulate phenolic acids or tanshinones biosynthesis. Then, we analyzed their function by over expression in hairy roots and identified their target genes which involved in phenolic acids and tanshinones biosynthesis pathways. In addition, we found the synergistic regulatory mechanism of SmbHLH7 and SmMYB39 had three levels: SmMYB39 can competitively bind key genes of phenolic acids or tanshinones metabolic pathway with SmbHLH7, negative regulator SmMYB39 directly inhibits SmbHLH7 transcription, SmMYB39 and other MYB competitively interact with SmbHLH7, preventing the formation of the positive-regulating MBW complex.

Subject Area生理生态学
MOST Discipline Catalogue理学::生态学
Pages106
Language中文
Document Type学位论文
Identifierhttp://ir.iswc.ac.cn/handle/361005/8775
Collection水保所2018--2019届毕业生论文
Recommended Citation
GB/T 7714
邢丙聪. 丹参转录因子bHLH7与MYB39互作调控酚酸类及丹参酮类物质代谢分子机制研究[D]. 北京. 中国科学院大学,2019.
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