本文選題：丹參 + MYB轉(zhuǎn)錄因子��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：丹參(SalviamiltiorrhizaBunge)是藥用模式植物,其干燥根莖可用于預(yù)防和治療多種疾病,例如冠心病、動脈粥樣硬化和心絞痛等。酚酸和丹參酮是丹參的兩大主要活性成分。近五年來,使用現(xiàn)代生物技術(shù)手段闡明丹參的次生代謝途徑、調(diào)控活性成分的生物合成吸引了更多關(guān)注。本研究通過在丹參毛狀根中過表達(dá)SmMYB36,嘗試探討其對活性物質(zhì)積累的調(diào)控機(jī)制。具體如下:1.從丹參中克隆了 SmMYB36基因,通過序列比對和二級結(jié)構(gòu)分析確定SmMYB36是典型的R2R3-MYB轉(zhuǎn)錄因子。使用雙向BLAST方法,找到了SmMYB3 在不同物種中的直系同源基因,其在擬南芥中的直系同源基因為AtMYB23。使用進(jìn)化分析和二級結(jié)構(gòu)分析證明SmMYB36在進(jìn)化上十分獨特。2.使用基因槍方法,將載體pA7-GFP-SmMYB36和pA7-GFP分別轉(zhuǎn)化洋蔥表皮細(xì)胞,觀察SmMYB36的定位情況�？蛰d對照的綠色熒光出現(xiàn)在細(xì)胞核和細(xì)胞質(zhì);SmMYB36的綠色熒光,在細(xì)胞核和細(xì)胞質(zhì)中均有發(fā)現(xiàn),細(xì)胞核中的綠色熒光十分明亮,細(xì)胞質(zhì)中的綠色熒光呈彌散狀態(tài)。3.構(gòu)建了過表達(dá)載體pK7WG2R-SmMYB36,使用發(fā)根農(nóng)桿菌ATCC15834誘導(dǎo)丹參葉片,成功誘導(dǎo)出過表達(dá)SmMYB36的毛狀根。對照毛狀根呈淡黃色,過表達(dá)SmMYB36的毛狀根呈磚紅色。使用高效液相色譜法、氣相色譜法和生理方法測定毛狀根中代謝物質(zhì)的含量,發(fā)現(xiàn)過表達(dá)SmMYB36顯著(P0.05)抑制了迷迭香酸、丹酚酸B、總酚、總黃酮和油酸的積累,顯著(P0.05)促進(jìn)了二氫丹參酮Ⅰ、隱丹參酮、丹參酮Ⅰ、丹參酮ⅡA和亞油酸積累。4.使用熒光定量PCR分析過表達(dá)SmMYB36毛狀根中次生代謝途徑基因表達(dá)量的變化,發(fā)現(xiàn)基因表達(dá)量變化與代謝物含量變化一致,過表達(dá)SmMYB36使苯丙烷衍生途徑基因(PAL1、C4H1和4CL2)和酪氨酸衍生途徑基因(TAT1)的表達(dá)顯著(P0.05)降低,使磷酸赤蘚糖醇途徑的基因(DXS1、DXS2、DXR MCT、MDS、HDS、CMK和HDR1)和下游合成途徑的基因(GGPPS1、CPS1、KSL1和CYP76AHI)表達(dá)量有所增加。5.構(gòu)建了原核表達(dá)載體pET32a-SmMYB36并轉(zhuǎn)化大腸桿菌表達(dá)菌株BL21進(jìn)行蛋白表達(dá)。使用HIS標(biāo)簽純化樹脂進(jìn)行蛋白純化并進(jìn)行western雜交驗證,結(jié)果顯示得到目的蛋白。使用丹參基因組數(shù)據(jù)庫獲得代謝途徑基因的啟動子區(qū),使用PlantCARE軟件分析尋找啟動子區(qū)是否存在MYB響應(yīng)元件(MBS1、MBS2、MBS3、MRE、MBSⅠ和MBS Ⅱ)。根據(jù)元件序列設(shè)計探針,使用凝膠電泳遷移法探索SmMYB36能否和探針進(jìn)行體外互作。結(jié)果表明SmMYB36能夠與探針MBS1、MBS Ⅰ和MBS Ⅱ進(jìn)行體外互作。
[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，

本文編號：1819434