【作者】 柳忠玉；

【导师】 赵树进；

【作者基本信息】 华南理工大学 ， 发酵工程， 2012， 博士

【摘要】 白藜芦醇是植物在长期进化过程中为增强自身对不良环境（如病原菌入侵、紫外线辐射等）而合成的植保素。研究报道白藜芦醇具有广泛的药理作用，包括抗肿瘤、保护心血管系统、延缓衰老等。白藜芦醇合酶是白藜芦醇生物合成途径中的关键酶之一，该酶与查尔酮合酶催化相同的底物。已有的研究表明白藜芦醇合酶基因与查尔酮合酶基因可能在生物合成途径中相互影响和相互调节。尽管关于白藜芦醇合酶与查尔酮合酶基因的研究报道很多，但是来源于虎杖的白藜芦醇合酶和查尔酮合酶基因的研究鲜有报道。已有研究者利用同源克隆的方法从虎杖中分离了基因PcRS和PcPKS1，推测其分别编码白藜芦醇合酶和查尔酮合酶。但是这两个基因在植物体内的功能以及如何调控白藜芦醇生物合成还未见文献报道。本课题通过在转基因拟南芥和虎杖植株中过量表达PcRS基因以及RNA干涉抑制PcPKS1基因的表达这两种策略，来考察分析PcRS基因和PcPKS1基因对白藜芦醇生物合成的调控作用，这对虎杖白藜芦醇生物合成途径的深入了解有非常重要的意义。本文的主要研究结果如下：（1）首先考察了PcRS基因的组织表达特性，Northern blot分析表明该基因在虎杖根茎中特异表达，这种转录模式与HPLC测得的白藜芦醇在虎杖各组织中的分布模式一致，推测PcRS基因与白藜芦醇生物合成有关。（2）为了分析PcRS基因在植物体内的功能，构建CaMV355启动子驱动的PcRS过量表达载体pCAMBIA1380-35S-PcRS并转化拟南芥。经高效液相色谱分析和质谱鉴定，转基因拟南芥植株能产生新的特异成分白藜芦醇苷。进一步分析发现，虽然外源PcRS基因在拟南芥叶、花、茎中组成型表达，但是白藜芦醇苷含量分布具有组织特异性，其中生长4周的叶片中白藜芦醇苷的含量最高（183.73μg/g鲜重）。以上结果证明，PcRS基因在植物体内具有促进白藜芦醇合成的功能，但是白藜芦醇的合成积累可能受PcRS基因的表达与底物浓度共同调控。（3）为了考察转基因拟南芥植株产生白藜芦醇苷是否具有生物活性，测定转基因拟南芥产生的白藜芦醇苷对炭疽病原菌的抑制率，当白藜芦醇苷浓度为50μg/mL时，抑制率达到50%。进一步用离体叶片法鉴定转基因拟南芥对炭疽病的抗性，叶片坏死斑直径减少56-65%，坏死斑上的孢子数减少75-80%，表明转基因拟南芥对炭疽病抗性增强。以上结果表明，转基因拟南芥植株产生的白藜芦醇苷具有抗炭疽病原菌的生物活性，推测PcRS基因可能参与植物抗病性调节。（4）为了确认白藜芦醇苷在转基因拟南芥中的积累是否会影响其它次生代谢物的合成，进行了进一步研究。野生拟南芥种皮颜色为褐色，而转基因拟南芥的种皮变为黄色，转基因拟南芥种皮中单宁合成受到抑制。转基因拟南芥子叶颜色变浅，花青素含量显著减少46-50%，表明转基因拟南芥中的花青素合成受到抑制。以上结果表明，外源PcRS基因的导入引起转基因拟南芥植株自身的黄酮物质的代谢流受阻。而表达分析显示转基因拟南芥内源的查尔酮合酶基因AtCHS表达没有下调。推测这种表型的改变不是由于内源AtCHS基因的下调，而是由于外源PcRS与内源AtCHS竞争底物。（5）在验证了PcRS基因功能的基础上开展了利用过量表达PcRS基因的策略来调控虎杖中白藜芦醇生物合成的工作。首先以虎杖茎尖为转化受体，对影响转化效率的因素如对潮霉素的敏感性、农杆菌液浓度、浸染时间、共培养时间、预培养时间等进行考察，建立根癌农杆菌介导虎杖茎尖遗传转化体系。随后利用该转化体系，将PcRS过量表达载体导入虎杖中并成功获得转基因虎杖。转基因虎杖及其后代表型无显著变化。转基因虎杖中PcRS的表达量是对照植株的1.4～1.9倍；白藜芦醇苷含量是对照植株的1.2～1.9倍。以上结果表明PcRS基因过量表达促进了虎杖中白藜芦醇的生物合成。（6）PcRS基因过量表达在一定程度上能促进虎杖中白藜芦醇生物合成，但是其含量提高的幅度并不大，于是开展了利用RNA干涉抑制PcPKS1基因表达的策略来调控虎杖中白藜芦醇生物合成的工作。首先构建CaMV355启动子驱动的PcPKS1干涉载体（pRNAi-PcPKS1）。随后利用已建立的转化体系将pRNAi-PcPKS1导入虎杖中，并成功获得转基因虎杖。转基因株系中PcPKS1的表达量明显低于对照植株，表达量下调39-87%；白藜芦醇苷含量明显提高，是对照植株的2.5-4.1倍；同时总黄酮含量减少14-42%；槲皮素含量减少22-62%。表型观察发现，表达量下调最多的转基因株系B5及其后代的表型都发生明显变化，其茎杆颜色变浅，花青素含量显著减少52%。以上结果表明，PcPKS1基因的表达下调抑制了虎杖黄酮物质的合成，使代谢流偏向白藜芦醇的合成，从而在一定程度上促进了虎杖中白藜芦醇的生物合成。综上所述，本课题证明PcRS基因编码的酶在植物体内具有白藜芦醇合酶的功能。PcRS过量表达和PcPKS1基因RNA干涉都能促进虎杖中白藜芦醇的生物合成。更多还原

【Abstract】 Resveratrol is a phytoalexin accumulated in some plant species in response to fungalinfection or UV irradiation. Resveratrol have been tested to have significant pharmaceuticaleffects in prevention of cancers, heart diseases and neurodegenerative diseases. Resveratrolsynthase (RS), competing with chalcone synthase(CHS) for the same substrates, is the keyenzyme to synthesize resveratrol. Although RS and CHS genes have been well characterizedin many plant species, there are only a few descriptions about RS and CHS genes fromPolygonum cuspidatum Sieb.Recently, a resveratrol synthase gene (PcRS) and a chalcone synthase gene (PcPKS1)were isolated in P. cuspidatum, but there are only a few descriptions about the metabolicfunction of PcRS and PcPKS1in planta. In this study, to understand the regulation of PcRSand PcPKS1in the resveratrol biosynthesis, the PcRS gene was over-expressed in Arabidopsisand P. cuspidatum. On the other hand, we use RNAi technology to inhibite the geneexpression of PcPKS1in P. cuspidatum. The main results were as follows:（1）Northern blot analysis revealed that the accumulation of PcRS transcripts was mostabundant in rhizomes. This distribution corresponds well with the accumulation of theresveratrol, suggesting that PcRS might play an important role in resveratrol biosynthesis.（2）To understand the metabolic function of PcRS in planta, it was placed under thecontrol of the cauliflower mosaic virus (CaMV)35S promoter and overexpressed intransgenic Arabidopsis thaliana via Agrohacterium-mediated transfer methods. Transgenicplants accumulated a new compound, which was identified as piceid by high-pressure liquidchromatography (HPLC) and electrospray mass spectrometry (HPLC–ESI–MS). Nosignificant difference in PcRS transcript levels was observed in leaves, stems and roots, whilepiceid concentration was highest in the leaves of4-week-old Arabidopsis plants, up to183.73μg/g FW. We showed that overexpression of PcRS in transgenic Arabidopsis resultedin accumulation of piceid. But the successful accumulation of resveratrol is likely to bedependent upon a combination of sufficient PcRS levels and the availability of commonsubstrates. （3）Piceid was purified by preparative HPLC from transgenic lines and tested inagar-plate bioassays in order to assess its effectiveness against fungal. At the concentration of50μg/mL, hyphal growth was approximately reduced (50%). Results showed that themycelial growth of Colletotrichum higginsianum was greatly inhibited. The detached leavesof plants were used for spot inoculations.The lesion diameter of the transgenic Arabidopsisplants was56-65%smaller than that of the wild-type plants. Spore production of thetransgenic plants was75-80%less than the wild-type plants. Overexpression of PcRS intransgenic Arabidopsis caused restriction of C. higginsianum colonization by inhibition ofspore production, resulting in enhanced resistance against C. higginsianum.（4）We investigated whether the accumulation of piceid in transgenic Arabidopsis plantswould affect other secondary metabolite pools. The mature seed coats of wild-type plantswere brown, whereas the seeds of transgenic Arabidopsis appeared pale fawn. The transgeniclines had a remarkable decrease of46-50%in anthocyanin accumulation. These resultsindicate that both tannin and anthocyanin accumulation are decreased due to ectopicexpression of PcRS. Northern analysis showed that expression of endogenous Arabidopsischalcone synthase gene (AtCHS) in the transgenic lines was not decreased. These resultssuggest that the significant reduction in flavonoid levels in transgenic plants may be due tocompetition for the substrates, instead of suppression of endogenous AtCHS expression.（5）To understand the metabolic function of PcRS in P. cuspidatum, PcRS wasoverexpressed in transgenic P. cuspidatum. Prior to the transformation experiments, thefactors influencing A.tumefaciens-mediated transformation of P. cuspidatum were explored tooptimize the transformation system, which included sensitivity of shoot tips to hygromycin,preculture period, Agrobacterium concentration, infection time, co-cultivation period andpre-culturing time. Finally, a system of high efficiency of genetic transformation andregeneration of P. cuspidatum was established. The plant expression vectorspCAMBIA1380-35S-PcRS was introduced into P. cuspidatum. No significant phenotypicdifference was observed in transgenic plants and the second asexual multiplicationgeneration. Nouthern blot exhibited a increase expression in all positive lines and expressionof PcRS were1.4to1.9times as control. Transgenic P. cuspidatum produced1.2to1.9-foldhigher piceid than control plant. These results suggest that overexpression of PcRS activated resveratrol biosynthesis pathway in P. cuspidatum.（6）To understand the metabolic function of PcPKS1, the plant expression vectorpRNAi-PcPKS1were introduced into Polygonum cuspidatum. The transgenic lines hadremarkable decrease of39-87%in PcPKS1transcript levels,14-42%in flavonoidaccumulation,22-62%in quercetin accumulation. But transgenic P. cuspidatum produced2.5to4.1-fold higher piceid than control plant. The transgenic line B5had a remarkable decreaseof52%in anthocyanin accumulation, with altered stem color. These results suggest that theflavonoid content can be reduced by restrained PcPKS1gene expression using RNAi method,at the same time, resveratrol biosynthesis pathway was activated.In this study, the effectiveness of PcRS in improving stilbene accumulation in foreignspecies has been proved. Our results suggest that PcRS overexpression and PcPKS1geneexpression using RNAi activated resveratrol biosynthesis pathway.更多还原