本文關(guān)鍵詞： 白木香 倍半萜 萜類合成酶 酶學性質(zhì)　出處：《廣東藥科大學》2017年碩士論文　論文類型：學位論文

【摘要】：國產(chǎn)沉香(agarwood、Chinese eaglewood)為瑞香科植物白木香含有黑色樹脂的木質(zhì)部。白木香(Aquilaria sinensis(Lour.)Gilg)是我國國產(chǎn)沉香的唯一植物資源,健康生長的白木香木質(zhì)柔軟,白色,沒有香氣,只有在受到外界傷害,如自然惡劣條件下的風折、雷擊、蟲蛀等刺激和人為條件下的砍傷、接菌等傷害后才能在傷口處分泌黑色樹脂,進而形成沉香。沉香的化學成分主要為倍半萜類、2-(2-苯乙基)色酮類和芳香族類化合物。人工誘導的沉香與天然沉香的化學組成差異較大,天然沉香中主要成分為倍半萜類化合物和2-(2-苯乙基)色酮類化合物,且含量大致相同;人工沉香中主要為2-(2-苯乙基)色酮類物質(zhì)。倍半萜類化合物是白木香處于逆境脅迫狀況下時產(chǎn)生的防御物質(zhì),也是沉香的特征性成分和藥理活性成分。篩選白木香倍半萜類成分的誘導子和萜類合成途徑中關(guān)鍵限速酶基因的克隆表達分析,對于闡明沉香(樹脂)形成的分子機制、人工誘導結(jié)香過程的調(diào)控和提高人工沉香品質(zhì)具有重要意義。課題組前期野外白木香促香試驗研究證明甲酸結(jié)合白木香內(nèi)生真菌A13為高效的促香誘導子,本文此基礎(chǔ)上,以白木香離體枝條模型為基礎(chǔ),用誘導子(甲酸、水楊酸、茉莉酸甲酯和白木香內(nèi)生真菌A13)單獨誘導或做種誘導子綜合刺激誘導,篩選高效、專一誘導白木香萜類合成酶基因的表達和倍半萜類成分形成的誘導子;采用GC-MS聯(lián)用技術(shù)對離體枝條進行次級代謝產(chǎn)物分析,檢測并鑒定倍半萜類成分;運用熒光定量PCR技術(shù)分析萜類合成酶基因HMGR和DXS的表達水平,結(jié)合GC-MS檢測結(jié)果,綜合評價誘導子的誘導效果,最終確定“1%茉莉酸甲酯加10%A13”和“1%茉莉酸甲酯加15%A13”的誘導子組合可以選擇性地誘導白木香產(chǎn)生倍半萜類成分。在前期轉(zhuǎn)錄組測序的基礎(chǔ)上,從化學傷害白木香結(jié)香部位cDNA中克隆得到萜類合成途徑中的關(guān)鍵限速酶基因HMGR,其序列全長為1779 bp。將基因片段插入pET22b中構(gòu)建重組表達載體,在大腸桿菌中進行表達,經(jīng)親和層析純化后獲得具有生物活性的3-羥基-3-甲基戊二酰輔酶A還原酶(HMGR),經(jīng)SDS-PAGE電泳分析,Western-blot鑒定,HMGR蛋白以包涵體形式表達,其蛋白分子量大小為68 kD。HMGR蛋白的最佳誘導條件為IPTG濃度為0.5 mM,37℃條件下誘導4 h。HMGR蛋白包涵體經(jīng)透析復(fù)性后以HMG-CoA為底物進行酶學性質(zhì)分析,最終確定在35℃,pH7.0,HMG-CoA濃度為0.1mM時反應(yīng)4 min為最佳酶促反應(yīng)條件。本文結(jié)合白木香次生代謝產(chǎn)物分析與萜類生物合成酶基因的表達水平研究,可全面評價白木香倍半萜類誘導子的誘導效果,同時對萜類合成酶基因的克隆表達研究對闡明沉香倍半萜類化合物的生物合成機制,提高人工誘導白木香中倍半萜類物質(zhì)含量奠定基礎(chǔ),為人工誘導白木香結(jié)香過程的代謝調(diào)控,優(yōu)化人工造香技術(shù)提供科學依據(jù)。
[Abstract]:Agaarwood, made in China. Chinese eaglewoodis the xylem of the family Daphaceae, which contains black resin. Gilg is the only plant resource in China. Healthy growth of white wood soft, white, no aroma, only by external damage, such as natural adverse conditions under wind break, lightning strike, moth and other stimuli and human conditions cut. The black resin could be secreted at the wound after the injury of bacteria, and the chemical composition of Acanthopsis sinensis was mainly sesquiterpenoids. The chemical composition of aromatics induced by artificial aromatics was different from that of natural ones. Sesquiterpenoids and 2-diphenylethyl) chromatic ketones are the main components in the natural Amorpha sinensis, and the contents of them are about the same. The sesquiterpenoids are the defense substances produced in the condition of stress stress in Artemisia sinensis, which is mainly composed of 2-diphenylethyl) chromoketones and sesquiterpenoids. It is also the characteristic component and pharmacological active component of Acanthopsis sinensis. Screening the inducer of sesquiterpenes and the cloning and expression of key rate-limiting enzyme gene in the synthesis pathway of sesquiterpenes. For elucidating the molecular mechanism of the formation of the resin. It is of great significance to regulate the process of artificial induction and to improve the quality of artificial aroma. The field experiments of aromaena aromatica showed that formic acid combined with the endophytic fungus A13 was an effective inducer. In this paper, based on the in vitro shoot model, the elicitor (formic acid, salicylic acid, methyl jasmonate and endophytic fungus A13) was used to induce the induction of seed elicitor. To screen highly efficient and specifically induce the expression of terpene synthase gene and the elicitor of sesquiterpene components. The secondary metabolites of isolated branches were analyzed by GC-MS, and sesquiterpenes were detected and identified. The expression levels of HMGR and DXS of terpene synthase genes were analyzed by fluorescence quantitative PCR. Combined with the results of GC-MS detection, the induction effect of the inducers was evaluated synthetically. "1% methyl jasmonate plus 103" and "1% methyl jasmonate add 153" The elicitor combination can selectively induce sesquiterpenes to produce sesquiterpenes on the basis of pre-transcriptional sequencing. The key rate-limiting enzyme gene HMGR in terpene synthesis pathway was cloned from cDNA of chemical injury site. The gene fragment was inserted into pET22b to construct the recombinant expression vector, which was expressed in Escherichia coli. The bioactive 3-hydroxy-3-methyl-glutaryl coA reductase (HMGRN) was purified by affinity chromatography and analyzed by SDS-PAGE electrophoresis. HMGR protein was expressed as inclusion body by Western-blot. The optimal induction conditions for the protein with molecular weight of 68 kD.HMGR were as follows: the concentration of IPTG was 0.5 mm. The inclusion bodies of HMGR protein were induced at 37 鈩，

本文編號：1484781