白木香倍半萜诱导子的筛选与萜类合成酶基因的克隆表达分析

发布时间：2018-02-02 14:47

  本文关键词： 白木香 倍半萜 萜类合成酶 酶学性质　出处：《广东药科大学》2017年硕士论文　论文类型：学位论文

【摘要】：国产沉香(agarwood、Chinese eaglewood)为瑞香科植物白木香含有黑色树脂的木质部。白木香(Aquilaria sinensis(Lour.)Gilg)是我国国产沉香的唯一植物资源,健康生长的白木香木质柔软,白色,没有香气,只有在受到外界伤害,如自然恶劣条件下的风折、雷击、虫蛀等刺激和人为条件下的砍伤、接菌等伤害后才能在伤口处分泌黑色树脂,进而形成沉香。沉香的化学成分主要为倍半萜类、2-(2-苯乙基)色酮类和芳香族类化合物。人工诱导的沉香与天然沉香的化学组成差异较大,天然沉香中主要成分为倍半萜类化合物和2-(2-苯乙基)色酮类化合物,且含量大致相同;人工沉香中主要为2-(2-苯乙基)色酮类物质。倍半萜类化合物是白木香处于逆境胁迫状况下时产生的防御物质,也是沉香的特征性成分和药理活性成分。筛选白木香倍半萜类成分的诱导子和萜类合成途径中关键限速酶基因的克隆表达分析,对于阐明沉香(树脂)形成的分子机制、人工诱导结香过程的调控和提高人工沉香品质具有重要意义。课题组前期野外白木香促香试验研究证明甲酸结合白木香内生真菌A13为高效的促香诱导子,本文此基础上,以白木香离体枝条模型为基础,用诱导子(甲酸、水杨酸、茉莉酸甲酯和白木香内生真菌A13)单独诱导或做种诱导子综合刺激诱导,筛选高效、专一诱导白木香萜类合成酶基因的表达和倍半萜类成分形成的诱导子;采用GC-MS联用技术对离体枝条进行次级代谢产物分析,检测并鉴定倍半萜类成分;运用荧光定量PCR技术分析萜类合成酶基因HMGR和DXS的表达水平,结合GC-MS检测结果,综合评价诱导子的诱导效果,最终确定“1%茉莉酸甲酯加10%A13”和“1%茉莉酸甲酯加15%A13”的诱导子组合可以选择性地诱导白木香产生倍半萜类成分。在前期转录组测序的基础上,从化学伤害白木香结香部位cDNA中克隆得到萜类合成途径中的关键限速酶基因HMGR,其序列全长为1779 bp。将基因片段插入pET22b中构建重组表达载体,在大肠杆菌中进行表达,经亲和层析纯化后获得具有生物活性的3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR),经SDS-PAGE电泳分析,Western-blot鉴定,HMGR蛋白以包涵体形式表达,其蛋白分子量大小为68 kD。HMGR蛋白的最佳诱导条件为IPTG浓度为0.5 mM,37℃条件下诱导4 h。HMGR蛋白包涵体经透析复性后以HMG-CoA为底物进行酶学性质分析,最终确定在35℃,pH7.0,HMG-CoA浓度为0.1mM时反应4 min为最佳酶促反应条件。本文结合白木香次生代谢产物分析与萜类生物合成酶基因的表达水平研究,可全面评价白木香倍半萜类诱导子的诱导效果,同时对萜类合成酶基因的克隆表达研究对阐明沉香倍半萜类化合物的生物合成机制,提高人工诱导白木香中倍半萜类物质含量奠定基础,为人工诱导白木香结香过程的代谢调控,优化人工造香技术提供科学依据。
[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，

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