丹参SmMYB36基因的功能鉴定与代谢调控机制研究

发布时间：2018-04-29 09:56

  本文选题：丹参 + MYB转录因子　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：丹参(SalviamiltiorrhizaBunge)是药用模式植物,其干燥根茎可用于预防和治疗多种疾病,例如冠心病、动脉粥样硬化和心绞痛等。酚酸和丹参酮是丹参的两大主要活性成分。近五年来,使用现代生物技术手段阐明丹参的次生代谢途径、调控活性成分的生物合成吸引了更多关注。本研究通过在丹参毛状根中过表达SmMYB36,尝试探讨其对活性物质积累的调控机制。具体如下:1.从丹参中克隆了 SmMYB36基因,通过序列比对和二级结构分析确定SmMYB36是典型的R2R3-MYB转录因子。使用双向BLAST方法,找到了SmMYB3 在不同物种中的直系同源基因,其在拟南芥中的直系同源基因为AtMYB23。使用进化分析和二级结构分析证明SmMYB36在进化上十分独特。2.使用基因枪方法,将载体pA7-GFP-SmMYB36和pA7-GFP分别转化洋葱表皮细胞,观察SmMYB36的定位情况。空载对照的绿色荧光出现在细胞核和细胞质;SmMYB36的绿色荧光,在细胞核和细胞质中均有发现,细胞核中的绿色荧光十分明亮,细胞质中的绿色荧光呈弥散状态。3.构建了过表达载体pK7WG2R-SmMYB36,使用发根农杆菌ATCC15834诱导丹参叶片,成功诱导出过表达SmMYB36的毛状根。对照毛状根呈淡黄色,过表达SmMYB36的毛状根呈砖红色。使用高效液相色谱法、气相色谱法和生理方法测定毛状根中代谢物质的含量,发现过表达SmMYB36显著(P0.05)抑制了迷迭香酸、丹酚酸B、总酚、总黄酮和油酸的积累,显著(P0.05)促进了二氢丹参酮Ⅰ、隐丹参酮、丹参酮Ⅰ、丹参酮ⅡA和亚油酸积累。4.使用荧光定量PCR分析过表达SmMYB36毛状根中次生代谢途径基因表达量的变化,发现基因表达量变化与代谢物含量变化一致,过表达SmMYB36使苯丙烷衍生途径基因(PAL1、C4H1和4CL2)和酪氨酸衍生途径基因(TAT1)的表达显著(P0.05)降低,使磷酸赤藓糖醇途径的基因(DXS1、DXS2、DXR MCT、MDS、HDS、CMK和HDR1)和下游合成途径的基因(GGPPS1、CPS1、KSL1和CYP76AHI)表达量有所增加。5.构建了原核表达载体pET32a-SmMYB36并转化大肠杆菌表达菌株BL21进行蛋白表达。使用HIS标签纯化树脂进行蛋白纯化并进行western杂交验证,结果显示得到目的蛋白。使用丹参基因组数据库获得代谢途径基因的启动子区,使用PlantCARE软件分析寻找启动子区是否存在MYB响应元件(MBS1、MBS2、MBS3、MRE、MBSⅠ和MBS Ⅱ)。根据元件序列设计探针,使用凝胶电泳迁移法探索SmMYB36能否和探针进行体外互作。结果表明SmMYB36能够与探针MBS1、MBS Ⅰ和MBS Ⅱ进行体外互作。
[Abstract]:Salvia miltiorrhizae Bungeis is a medicinal model plant, its dry rhizome can be used to prevent and treat many diseases, such as coronary heart disease, atherosclerosis and angina pectoris. Phenolic acid and tanshinone are two main active components of Salvia miltiorrhiza. In recent five years, using modern biotechnology to elucidate the secondary metabolic pathway of Salvia miltiorrhiza and regulate the biosynthesis of active components has attracted more and more attention. In this study, SmMYB36 was overexpressed in hairy roots of Salvia miltiorrhiza to explore its regulatory mechanism on the accumulation of active substances. The details are as follows: 1. SmMYB36 gene was cloned from Salvia miltiorrhiza. Sequence alignment and secondary structure analysis confirmed that SmMYB36 is a typical R2R3-MYB transcription factor. The direct homologous gene of SmMYB3 in different species was found by using bidirectional BLAST method. The direct homology of SmMYB3 in Arabidopsis was AtMYB23. Evolutionary analysis and secondary structure analysis show that SmMYB36 is unique in evolution. The vector pA7-GFP-SmMYB36 and pA7-GFP were transformed into onion epidermal cells by gene gun method to observe the localization of SmMYB36. The green fluorescence of the blank control appeared in the nucleus and cytoplasm of SmMYB36. It was found that the green fluorescence in the nucleus and cytoplasm was very bright, and the green fluorescence in the cytoplasm was diffuse. The overexpression vector pK7WG2R-SmMYB36 was constructed. The hairy roots of salvia miltiorrhiza were induced by Agrobacterium tumefaciens ATCC15834. The hairy root of control group was light yellow, and the hairy root with SmMYB36 overexpression was brick red. High performance liquid chromatography (HPLC), gas chromatography (GC) and physiological methods were used to determine the contents of metabolites in hairy roots. It was found that overexpression of SmMYB36 significantly inhibited the accumulation of rosemary acid, Salvianolic acid B, total phenol, total flavonoids and oleic acid. P0.05) promoted the accumulation of dihydrotanshinone 鈪，

本文编号：1819434