【作者】 蒋科技；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 植物次生代谢产物是一大类的小分子化合物,在植物体中的基础含量一般都很低。然而在生物和非生物的压力胁迫下,或者在一定的诱导条件下,次生代谢产物的产量通常有一定的提高。近年来,为了提高特定的次生代谢产物的含量,代谢工程被广泛应用。随着次生代谢产物生物合成途径中已克隆基因的增多以及代谢途径的进一步阐明,已经有一些行之有效的方法取得了令人欣喜的进展。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、乙烯以及茉莉酸酮酯(JAs)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,茉莉酸酮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。此外,茉莉酸酮酯在植物发育的各个阶段也具有重大的意义。在植物的茉莉酸生物合成途径中,丙二烯氧化物环化酶(AOC,allene oxidecycalse,EC 5.3.99.6)和丙二烯氧化物合成酶(AOS,allene oxide synthase,EC4.2.1.9)的催化是最重要的酶促步骤。为了研究药用植物茉莉酸合成途径基因的功能以及它们在次生代谢工程中的应用价值,选取了莨菪和金银花为研究对象。莨宕是一种重要的药用植物,会产生一类受甲基茉莉酸诱导的色胺类生物碱。我们从中克隆了JA合成途径的基因AOC和AOS,并把AOC基因转入烟草验证了其对次生代谢产物(尼古丁)合成的促进作用。金银花是一种常用的中草药,含有多种次生代谢产物,其中绿原酸是最重要的活性成分,也是衡量金银花质量的指标化合物,我们从中克隆了AOS基因,并发现其组织表达同绿原酸的组织分布有一定的相似性。1)莨宕丙二烯氧化物环化酶基因(HnAOC)丙二烯氧化物环化酶(AOC)的催化作用是JA合成途径中最关键的步骤。采用RACE的方法,从莨菪中克隆了AOC基因(HnAOC,GenBank登录号:AY708383),该基因与其他物种中的AOC基因具有较高的同源性。其cDNA全长为1044 bp,包括5’和3’非翻译区(UTR)、polyA尾和一个长747 bp的开放阅读框(ORF)。HnAOC基因组内含有两个内含子,分别为94 bp和402 bp。HnAOC基因编码的蛋白质共有248个氨基酸,分子量为27.0 kDa,理论等电点8.74。Southern杂交结果显示HnAOC在莨菪中是一个多拷贝基因;RT-PCR分析结果表明,HnAOC基因可被多种不同的诱导因素所诱导表达,从而可以借此调控AOC基因的表达和JA合成。2)莨宕丙二烯氧化物合成酶基因(HnAOS)丙二烯氧化物合成酶(AOS)是JA合成途径中的第一个特异性的酶。采用RACE的方法,从莨菪中克隆了AOS基因(HnAOS,GenBank登录号:EF532599),其cDNA全长为1652 bp,包括5’和3’非翻译区(UTR),polyA尾和一个长1485 bp的开放阅读框(ORF),基因组内不含有内含子。HnAOS基因编码的蛋白质含494个氨基酸,分子量为55.8 kDa,理论等电点8.82,属于细胞色素P450大家族。实时荧光定量PCR分析结果显示,在正常生长的植株中,HnAOS基因在茎中表达水平最高。另外,HnAOS基因受伤、甲基茉莉酸的诱导而上调表达,直接的甲基茉莉酸处理比伤诱导的效果在时间上出现得更早,从而佐证了伤诱导的作用是通过茉莉酸合成途径来实现的。3)转基因烟草中HnAOC的过量表达提高了其抗逆性及尼古丁含量丙二烯氧化物环化酶的催化是植物JA合成途径中最关键的步骤,而且烟草中尼古丁的代谢受JA的诱导,为了验证JA生物合成途径的基因工程能否使受其诱导的次生代谢产物的含量增加,因此我们将最莨菪的AOC基因转入烟草,以尼古丁含量为代表来验证HnAOC过量表达对次生代谢产物含量的影响。RT-PCR分析结果显示,在转基因烟草中HnAOC均有表达,而烟草AOC基因的表达也增强了;电导率测定结果显示,转基因烟草的叶片在低温条件下离子泄漏程度低于野生型烟草,表现出对低温伤害的耐受性。实时荧光定量PCR(RT-QPCR)结果表明,转基因烟草中尼古丁合成途径的关键酶基因PMT和QPT的表达水平也大幅度提高,HPLC分析结果显示其尼古丁含量也相应提高了。因而,本研究证明了JA生物合成途径的基因操作可以代替繁琐的外源激素处理来提高目的产物的含量,从而为生产受JA诱导的次生代谢产物提供了一个有效的基因工程策略。4)金银花丙二烯氧化物合成酶基因(LjAOS)金银花是一种被广泛应用的重要的中草药材料,具有清热解毒,消炎杀菌的功效。绿原酸是金银花的主要功效成分,也是金银花质量监控的指标性化合物,它的含量在不同的组织和花周期中差异较大,其中在花蕾中的含量最高。对于药农和制药厂家来说,药材的质量监控是非常关键的问题,因此金银花采收部位的绿原酸含量备受关注。JA作为一类调控植物发育和次生代谢的植物激素,可能在其中起着重要的作用。在JA的生物合成途径中,AOS是第一个特异性的酶,属于细胞色素P450大家族。为了研究JA合成途径的遗传操作是否可以对绿原酸的合成进行调控,我们首先从金银花中克隆出来一个全新的AOS基因(LjAOS,GenBank登录号:DQ303120)。其cDNA全长为1942 bp,包括5’和3’非翻译区(UTR),polyA尾和一个长1584 bp的开放阅读框(ORF)。LjAOS基因编码的蛋白质含527个氨基酸,分子量为59.2 kDa,理论等电点为8.41,其基因组中不含有内含子。多重比对表明LjAOS蛋白与其他的AOS蛋白具有较高的同源性,LjAOS蛋白序列中含有AOS家族应有的保守氨基酸残基。Southern杂交结果显示LjAOS基因在金银花中为多拷贝基因;有趣的是,RT-QPCR结果显示LjAOS基因在各个组织中表达量也是在花蕾中最高,这与绿原酸的分布规律具有一定的相似性,揭示了JA的生物合成可能与花的发育以及绿原酸的合成有关。本研究为提高金银花中绿原酸含量提供了一个可供试验的靶点。综上所述,莨菪和金银花JA合成途径关键酶基因的研究使我们对JA生物合成途径和其在上述两种药用植物中的调控作用有了一定的了解,对于进一步阐明植物JA合成途径及其作用机制和进化有一定的帮助。同时,对转基因烟草抗逆性能和尼古丁含量的研究将有利于JA合成途径基因在植物次生代谢工程中的应用。更多还原

【Abstract】 Plant secondary metabolites are a wide variety of low-molecular-weight compounds whose productions are often enhanced in response to both biotic and abiotic stresses. Nevertheless,most of the important secondary metabolites are of low content,or can only be produced with elicitation.Recently,metabolic engineering has been widely applied in order to achieve higher yields of specific metabolites.With the increasing number of cloned genes involved in biosynthesis and the expanding investigation of biosynthesis pathway,some effective approaches have showed encouraging progresses.Under biotic or abiotic stress,plants produce increased amounts of hormones such as abscisic acid(Sasaki et al.),SA(salicylic acid),ethylene and jasmonate(JA).These hormones may interact with one another in regulating stress signaling,plant stress tolerance and secondary metabolism.Many of the responses are mediated by a class of hormones named as JAs.JAs play significant roles in the biosynthesis of many plant secondary metabolites as well as in distinct developmental stages.In jasmonate biosynthetic pathway of plants,allene oxide synthase(AOS,EC 4.2.1.9)and allene oxide cyclase(AOC,EC 5.3.99.6)catalyzes the most important steps.Hyoscyamus niger is a medicinal plant which produces a class of jasmonate-responsive pharmaceutical secondary compounds named as tropane alkaloids.Honeysuckle(Lonicera spp.,named "jin-yin-hua" in Chinese,also known as Japanese Honeysuckle,whose flowers enjoy name of Flos Lonicerae as medicinal part),is an important crude herb frequently used in Chinese medicines,containing various metabolites with chlorogenic acid(CA,the major important active ingredient) as the indicator compound to characterize the quality of this herb.1)Allene oxide cyclase gene from H.nigerA novel cDNA encoding for allene oxide cyclase from H.niger,named HnAOC (GenBank accession;AY708383.)was cloned by RACE,showing highly homologous to the AOCs of other species.The full-length cDNA of HnAOC was 1044 bp with 5’ and 3’ UTR,polyA tail and a 747-bp ORE The comparison between the flall-length cDNA and the genomic DNA of HnAOC revealed that the genomic DNA contained two introns,one was 94 bp and the other one was 402 bp.The putative protein was 248 AA(amino acid)with a molecular weight of 27.0 kDa and a pI of 8.74.Southern blot analysis showed that it was a multi-copy gene.RT-PCR analysis revealed that the expression of HnAOC could be regulated by various stresses and elicitors,indicating the alternative ways to regulate AOC expression and jasmonate biosynthesis.2)Allene oxide synthase gene from H.nigerA full-length cDNA encoding allene oxide synthase,the first commit step enzyme in jasmonate biosynthesis pathway,which was named as HnAOS(GenBank accession; EF532599)was cloned by RACE.The full-length HnAOS was 1652 bp with 5’-UTR, a 1485-bp ORF,3’-UTR(un-translated regions)and poly-A tail.The putative protein was 494 AA(amino acid)with a molecular weight of 55.8 kDa and a pI(Isoelectric Point)of 8.82.HnAOS was a novel member of the cytochrome P450(CYP74A) subfamily.There was no intron present in the genome DNA among HnAOS.Real-time quantitative PCR analysis showed that HnAOS mRNA mainly accumulated in stems. The expression level of HnAOS significantly increased with wounding or MeJA treatment,and MeJA showed the induction-effect earlier than wounding.Evidently,it was consist with that wounding acted its role by inducing JA biosynthesis.3)Overexpression of allene oxide cyclase from H.niger in transgenic tobaccos with the effects of both improving stress-tolerance and enhancing nicotine production.It was speculated that genetic engineering of jasmonate biosynthetic pathway might enhance the endogenous jasmonates concentrations thus stimulate the production of jasmonate-responsive secondary metabolites.The allene oxide cyclase catalyzed step is the most important one in jasmonate biosynthetic pathway of plants.Here the heterogenous AOC gene from H.niger was transformed into Nicotiana tabacum cv. Petit Havana to investigate the influence of HnAOC overexpression on the nicotine content as a testing mode.The results revealed that the overexpression of HnAOC in tobacco not only enhanced stress-tolerance but also stimulated nicotine biosynthesis. Therefore,it was validated that without the cost of extraneous hormones,genetic manipulation of jasmonate biosynthetic pathway genes could be another alternative approach in secondary metabolic engineering for the production of valuable jasmonate-responsive secondary metabolites. 4)Allene oxide synthase gene from Lonicera japonicaHoneysuckle(Lonicera sp.)is an important Chinese herbal medicinal plant whose flowers(named "Flos Lonicerae" as a medicinal material)are widely used as anti-inflammatory agent in oriental area since ancient times.The content of chlorogenic acids,which represents the quality of this medicinal material,varies according to different tissues and distinct stages of development.So it is significantly important for farmers and medicinal manufactures to control the quality of this material.Jasmonates(JAs)are a class of phytohormones which play a central role in plant defense responses as well as in distinct stages of plant development.In order to investigate whether genetic manipulation of jasmonates biosynthesis pathway could regulate the development of honeysuckle coupled with the corresponding content of chlorogenic acids,we firstly cloned a novel cDNA from Lonicera japonica Thunb., named LjAOS(GenBank accession;DQ303120),which was homologous to other AOSs.The full-length cDNA of LjAOS was 1942 bp with 5’ and 3’ UTR,poly-A tail and contained a 1584-bp ORF.The putative protein was 527 AA with a molecular weight of 59.2 kDa and a pI of 8.41.No intron was present in the genomic DNA. Southern blot analysis revealed that it was a multi-copy gene.Real-time quantitative PCR analysis showed that LjAOS mRNA accumulated the most abundantly in alabastrums,in which the content of chlorogenic acids was also the highest.It indicated the positive relationship between JAs biosynthesis and the levels of chlorogenic acids.The study of JAs biosynthesis pathway in Lonicera japonica would help to facilitate the production of this medicinal material.In conclusion,the study of JA biosynthetic pathway in H.niger and L.japonica presented a knowledge of JA biosynthetic pathway and its regulation in these two medicinal plants.The results also helped to further elucidate the mechanisms and evolution of JA biosynthetic pathway in planta.Simultaneously,the examination of stress-tolerance and nicotine production in transgenic tobaccos as a testing mode would greatly shed light on the application of JAs in plant secondary metabolic engineering.更多还原