【作者】 谷晓策；

【导师】 陈万生；

【作者基本信息】 第二军医大学 ， 生药学， 2011， 硕士

【摘要】 植物次生代谢产物是植物为了抵御外界刺激如微生物入侵与昆虫、食草动物伤害等胁迫而产生的化合物,如抗生素、生物碱、毒素等。许多次生代谢产物具有很好的药理活性,是中药和天然植物药的主要有效成分。在外界生物或非生物的压力胁迫下,一些植物激素,如脱落酸、水杨酸、乙烯以及茉莉酸甲酯(MeJA)等在植物中大量积累,通过相互作用,调控植物的信号传递、胁迫耐受以及次生代谢等过程,形成植物的应激系统。其中,茉莉酸甲酯在植物应激反应和次生代谢调控中起着至关重要的作用。目前,MeJA作为外源诱导子促进植物次生代谢物的过量积累,已得到广泛应用。本研究探索一种新的代谢工程策略,通过过量表达植物MeJA合成途径上的关键酶基因,提高内源MeJA的含量,进而刺激多种次生代谢产物的合成与积累。为了使这种新策略应用于多种植物,节省大量克隆内源基因的工作。本研究同时将内源和异源基因在植物中过量表达,考察是否达到同样的效果。在植物的茉莉酸甲酯生物合成途径中,丙二烯氧化物环化酶allene oxide cycalse(AOC)和茉莉酸羧甲基转运酶jamonic acid carboxyl methyltransferase(JMT)是重要的关键酶。本研究采用RACE方法,从丹参中克隆了AOC基因(SmAOC,GenBank登录号:HM156740),其cDNA全长为910bp,开放阅读框(ORF区)编码的蛋白质共有245个氨基酸。该基因与其他物种中的AOC基因具有较高的同源性,与水稻OsAOC的相似度为75%,与玉米ZmAOC的相似度为74%。器官组成型分析,SmAOC在丹参中根茎叶均有分布,叶中表达量最高。Southern杂交结果显示SmAOC在丹参中是一个低拷贝基因;RT-QPCR分析显示AOC基因表达可被多种不同的诱导因素,如:茉莉酸甲酯、紫外线照射、低温胁迫所诱导,而提高表达水平。原核表达SmAOC,SDS-PAGE电泳检测得到了预期表达的目的蛋白。耐盐胁迫试验表明,表达SmAOC蛋白的细菌比对照更耐受高盐环境。丹参中两大类活性成分主要是以丹参酮IIA为代表的脂溶性二萜醌类化合物和以丹参酚酸B为代表的水溶性化合物。在根癌农杆菌C58C1介导的丹参发根中分别过量表达MeJA合成途径中的关键酶:内源的丹参SmAOC,异源的莨菪HnAOC和拟南芥AtJMT,结果显示在各转基因事件中,上述两类化合物的含量基本上均比对照有不同程度提高。在所有转基因株系中,转SmAOC组的S-7株系丹参酮IIA提高最多,达到野生发根的16.6倍,干重含量从0.037mg/g提高到0.56mg/g;转AtJMT组的J-5株系隐丹参酮提高最多,达到野生发根的16.8倍,干重含量从0.027mg/g提高到0.46mg/g;转HnAOC组的H-11株系迷迭香酸提高最多,达到野生型对照的2.19倍,干重含量从1.32mg/g提高到2.89mg/g;转SmAOC组的S-1株系丹参酚酸B最多比野生型对照发根提高了78%,干重含量从10.73mg/g提高到19.07mg/g。转基因丹参发根中其他活性物质(丹参酮Ⅰ和二氢丹参酮)以及迷迭香酸合成途径的中间体的含量也有不同程度的提高。同时RT-QPCR技术检测了MeJA、迷迭香酸及萜类合成途径上基因的表达水平,结果显示,转基因发根中的这些合成途径上的相关基因均有相应地上调。本研究从多种转基因事件证明了, MeJA生物合成途径的基因操作可以同时对多个酶促反应或者多条代谢途径进行全局调控,从而提高植物体内多种次生代谢产物的含量,并且过量表达异源和内源基因能够产生同样的效果。通过遗传转化获得的次生代谢产物含量的提高是稳定且持久的,体现出比外源诱导更可靠更稳定可遗传的优越性,为生产受MeJA诱导的次生代谢产物提供了一个有效的基因工程新策略。更多还原

【Abstract】 Plant secondary metabolites are not necessary in normal growth, development and reproduction processes, but often play an important role in plant defense against herbivory and other interspecies defenses. They are a wide variety of low-molecular weight compounds which often have pharmacal activities. Jasmonic acid methyl ester (MeJA) is important signaling molecules in plants, especially in defense reactions to environmental stresses, such as mechanical wounding or pathogen attack.On account of this theorem, we are going to exploit an novel stratagem of secondary metabolic engineering, over express the key enzyme genes in MeJA biosynthetic pathway, to result in the increase of endogenous MeJA content,then , to stimulate the biosynthesis and accumulation of multiple secondary metabolites.Allene oxide cycalse(AOC) and jasmonic acid carboxyl methyltransferase(JMT)are key enzymes in MeJA synthetic pathway. First, we cloned the AOC gene from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) and designated it as SmAOC (GenBank accession: HM156740). The SmAOC cDNA has a total length of 910 bp with an open reading frame (ORF) of 738 bp, and is predicted to encode a protein of 245 amino acid residues, sharing high degree of homology with AOCs from other plants. A 123bp intron was present in the genomic DNA. Southern-blot analysis revealed that SmAOC was a low copy gene. Real-time fluorescent semiquantitative PCR (RT-QPCR) analysis revealed that SmAOC was preferentially expressed in leaves compared with other tissues (roots and stems). When S. miltiorrhiza seedlings were induced by abiotic stimuli such as MeJA and low temperature (4℃) , SmAOC expression was markedly increased within 24h, but not obviously by ultraviolet irradiation (UV). The full-length of ORF was inserted into prokaryotic expression vector pET32a, which was then transferred into E. coli BL21 (DE3). The recombinant pET32a-SmAOC in bacteria induced by IPTG and the expression of fusion protein was revealed by SDS-PAGE. Overproduction of recombinant SmAOC resulted in an increased tolerance to salinity in transgenic E. coli.S. miltiorrhiza contain two major classes of chemicals, a class of lipid-soluble diterpene quinone pigments, generally known as tanshinones, and a class of water-soluble phenolic acids including salvianolic acids B, rosmarinic acid (RA).SmAOC from Salvia miltiorrhiz, HnAOC from Hyoscyamus niger and AtJMT from Arabidopsis thaliana were overexpressed in S. miltiorrhiz hairy roots which were Inducted by Agrobacterium rhizogenes C58C1. In the three transgenic affairs, higher contents of secondary metabolites were obtained in different degrees with the maximum of TanshinoneⅡA 16.6-fold and Salvianolic acid B 78% over control. The contents of other active compounds(rosmarinic acid、tanshinoneⅠ、cryptotanshinone and dihydrotanshinone) in transgentic hairy roots increased in different degrees, and so did rosmarinci acid biosynthetic compounds. Simultaneously, the RT-QPCR result illustrated that the expression of intermediates in MeJA、rosmarinci acid and terpenoid biosynthetic pathway were up-regulated accordingly.This study revealed that overexpression of MeJA biosynthetic pathway genes resulted in higher yield of secondary metabolites. Moreover, the higher yield was herediable and more stable and persistent than exogenous induction. Therefore, it indicated that genetic manipulation of MeJA biosynthetic pathway genes could be an alternative approach in metabolic engineering for the production of valuable secondary metabolites.更多还原