【作者】 秦秋琳；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 苯丙氨酸代谢途径(phenylpropanoid pathway)是植物次生代谢产物合成的重要途径,其下游的分支途径主要分黄酮类合成途径和木质素合成途径,生成的苯丙烷类代谢产物,包括香豆素、黄酮醇、木质素、软木酯和其它的苯类化合物,作为植物抗毒素、UV保护因子、花和果实色素、细胞壁结构成分和信号传导分子等,在植物生长与发育中发挥了重要的作用,并且其中的黄酮醇等有效药用成分还被广泛地应用于医药、保健和人们的日常生活中,苯丙氨酸代谢途径不仅对于植物自身还是对于人类的意义都非同寻常,因此利用基因工程手段改造该条途径,改善次生代谢产物的合成具有重大的实际意义。银杏树是一种古老的珍贵树种,有植物界的“活化石”和“大熊猫”的美誉,是我国特产的植物资源。银杏中的有效活性成分—银杏黄酮是治疗心脑血管疾病的特效药物,并且银杏木也是我国特有的优良木材,价格贵,用途广泛。苯丙氨酸代谢途径对于银杏来说意义非凡,银杏中的黄酮类药用成分、木质素等都来源于苯丙氨酸代谢途径。目前对于银杏苯丙氨酸代谢途径的研究还只限于关键酶基因的克隆和初步分析,进一步探索和改造银杏中苯丙氨酸代谢途径是未来发展的方向。在利用基因工程改造植谢步骤,甚至整条代谢途径,从而能够从宏观上调控代谢流量,弥补了常规代谢工物复杂的代谢途径中,转录因子是一个新型的、强有力的工具,转录因子可以控制多个代程中单个关键酶基因作用的不足。目前对苯丙氨酸代谢途径的转录调控研究主要集中于玉米、矮牵牛、金鱼草和拟南芥等植物的花色素合成的调控,以及松树、杉树和桉树等木本植物中木质素合成途径的调控,而对于银杏中苯丙氨酸代谢途径的转录调控的研究还是空白。本文首次从银杏中克隆了与苯丙氨酸代谢途径和木质素合成途径相关的一个R2R3-MYB类转录因子基因,GbMYB1,并对其功能进行了研究。本文根据已报道的与苯丙氨酸途径相关的R2R3-MYB类转录因子的保守区段设计简并引物,利用RACE(rapid amplification of cDNA end)方法获得了1384bp的全长cDNA(命名为GbMYB1),GbMYB1编码由347个氨基酸组成的开放性阅读框(ORF)。Southern blot分析证明该基因在银杏中以单拷贝形式存在。该基因具有典型的R2R3-MYB类转录因子的特征,进化树分析及同源比对结果表明,该蛋白与白云杉(Picea glauca)中木质素合成相关转录因子PgMYB1及火炬松(Pinus taeda)中木质素合成相关转录因子PtMYB1的序列最为同源,进化关系最为相近,三者DNA结合域的氨基酸序列几乎完全一致,这三个转录因子与拟南芥中的AtMYB20、AtMYB43和AtMYB99转录因子在进化关系上也属于同一分支,并且通过序列比对发现除了DNA结合域外,这几个转录因子还存在一个特有的保守肽段,因此判断GbMYB1转录因子与这几个蛋白同属一个亚类,是一类新的R2R3-MYB类转录因子,它们也可能具有相似的功能。GbMYB1融合GFP的亚细胞定位研究表明,GbMYB1基因是核定位基因,其编码的蛋白具有明显的核定位能力,符合推测的蛋白中的核定位信号特征。该基因在银杏的木质形成器官根、主茎和主茎木质部中表达量最高,而在叶、树皮和果实中表达量较低,推测该基因可能与木质部的形成以及发育相关。为了进一步证明该蛋白的功能,将该基因构入了原核表达载体中进行表达,获得了纯化蛋白,体外结合实验表明,该蛋白能够与木质素调控相关顺式元件之一的ACelement结合。将该基因构建到植物表达载体中并转化拟南芥,发现过量表达GbMYB1基因的拟南芥转基因植株与对照植株相比,表型发生了很大的变化,转基因植株明显矮小,生长缓慢,生长周期较对照植株延长了2-3周左右。取转基因植株和对照植株的茎杆进行徒手切片,利用间苯三酚对切片进行染色发现,过量表达GbMYB1基因的转基因植株的木质部细胞个体较小,排列紧密,某些细胞壁的厚度较对照植株厚,染色较深,由此得知在拟南芥中过量表达GbMYB1后能够促进拟南芥植株木质部的形成,因此推测转基因植株中木质素的含量高于对照植株,这进一步证明了GbMYB1基因参与了木质素代谢途径的调控。为了进一步研究GbMYB1转录因子对木质素合成途径的调控,对转基因植株中木质素合成途径中的关键酶基因进行了定量分析,发现转基因植株中木质素代谢途径重要基因CADc、CADd(肉桂酰乙醇脱氢酶)和C3H(香豆酰-3-羟化酶)的表达量明显增加,其中CADc的增加倍数最大,达到8倍。途径中其他基因的表达量也略有增加。此外还对苯丙氨酸代谢途径中若干关键酶进行了定量PCR检测,发现途径中第一步的限速酶基因PAL2(苯丙氨酸解胺酶)的表达量提高更多,达十几倍。证明GbMYB1转录因子可上调苯丙氨酸和木质素代谢途径中的某些关键酶基因。另一方面,还对拟南芥中与GbMYB1进化关系最近的几个转录因子的突变体进行了鉴定和分析。发现突变体植株没有生长滞后现象,某些株系甚至比野生型稍快,木质部细胞染色发现,某些株系的木质部细胞壁较野生型稍薄,细胞个体与野生型相似。对其苯丙氨酸和木质素代谢途径的关键酶基因的表达量也进行了分析,发现突变体植株中COMT、CADd基因的表达量明显降低,并且CHI、FLS等黄酮途径的关键酶基因也受下调。这与过量表达GbMYB1基因的转基因拟南芥中得到的结果相反,证明了GbMYB1基因与拟南芥中的同源基因在功能上的类似性,并进一步确定了GbMYB1转录因子对于苯丙氨酸代谢途径和木质素合成途径的调控作用。本实验对于银杏中苯丙氨酸代谢途径和木质素合成途径的转录调控研究迈出了第一步,为以后银杏中或其它物种中次生代谢途径的转录调控研究做出了铺垫,对将来利用基因工程方法改造银杏中或其他物种中苯丙氨酸代谢途径或木质素合成途径具有重要的参考意义。另外本论文还涉及了一部份东北红豆杉酵母单杂交文库的构建和筛选工作。试验成功构建了东北红豆杉均一化的酵母单杂交文库,并构建了五类与甲基茉莉酸和水杨酸应答相关的顺式元件鱼饵质粒,为在东北红豆杉中筛选与甲基茉莉酸和水杨酸应答相关的转录因子或与紫杉醇次生代谢相关的转录因子打下基础。本研究筛选获得两类转录因子基因,Homeodomain转录因子和Remorin转录因子。更多还原

【Abstract】 Phenylpropanoid pathway together with two primary branches of flavonoid metabolic pathway and lignin biosynthetic pathway is an important metabolic pathway in plants.The phenylpropanoid pathway,with its products can act as plant anti-toxin,pigmentation of flowers and fruits,components of cell wall and molecules of signal transduction,plays important roles in growth and development of plants. Furthermore,some active components in phenylpropanoid pathway,such as flavonoids,are also applied broadly in medicine and health of human beings. Therefore,modification of this pathway using metabolic engineering is important for application.Ginkgo biloba L.is ancient and precious in plant kingdom.It is usually called as the ’living fossil’.Because containing the active ingredients like flavonoids,G.biloba is thought to be valuable as medicine.On the other hand,G.biloba is also an expensive wood applied broadly.Phenylpropanoid pathway in G.biloba is very important because both the active ingredient flavonoids and lignin are all derived from this pathway.Analysis and modification of Phenylpropanoid pathway in G. biloba would have practical value in breeding and pharmaceutical research.The functions of an increasing number of plant transcription factors are being elucidating,and many of these factors have been found to impact flux through metabolic pathways.Because transcription factors,as opposed to most structural genes,tend to control multiple metabolic pathway steps,they have emerged as powerful tools for the manipulation of complex metabolic pathways in plants.In this thesis,a novel R2R3-MYB transcription factor gene GbMYB1,which involved in phenylpropanoid pathway and lignin biosynthetic pathway has been cloned and analyzed from G.biloba for the first time.Using RACE method,a 1384 bp full-length cDNA,designated as GbMYB1,was cloned from G.biloba,which contained an open reading frame encoding a deduced protein of 347 amino acids with a predicted molecular mass of 38.4-KDa and a calculated pI of 5.09.Sequence alignments with Arabidopsis and other plant species showed that GbMYB1 was a member of the R2R3 MYB transcription factor family. The predicted protein GbMYB1 had the highest homology with PgMYB1 from Picea glauca and PtMYB1 from Pinus taeda.Furthermore,the conserved domain R2R3 of GbMYB1 shared almost the complete similarity with the two proteins.Phylogenetic tree analysis showed that GbMYB1 was close to AtMYB20,AtMYB43 and AtMYB99 from Arabidopsis.Amino acid sequence alignment of these proteins revealed that all five proteins shared a small conserved motif adjacent to the R2R3 conserved domain.The presence of this small conserved motif may define a new R2R3 MYB subgroup,which possibly reflects the similar functions of its members.Subcellular location of GbMYB1 demonstrated that GbMYB1 was a nuclear-localized protein.Semi-quantitative RT-PCR analysis showed that GbMYB1 expressed preferentially in the wood forming tissues,such as stems and roots, especially in xylem,implying that GbMYB1 might be involved in the lignin biosynthetic pathway.EMSA of GbMYB1 protein analysis showed that GbMYB1 could bind to ACII element involved in lignin biosynthetic pathway.Transgenic Arabidopsis plants overexpressing GbMYB1 gene just grow to only half of the size of the control plants after several weeks’ growth,and the flowering time of transgenic lines overexpressing GbMYB1 was postponed greatly compared with the control plants.The life cycle of the transformed GbMYB1 transgenic lines was longer than that of the control plants for about 3-4 weeks.Moreover,transgenic lines overexpressing GbMYB1 showed the reduction to a certain extent in seed production. All the phenomena demonstrate that the transgenic Arabidopsis had dramatically lagging growth.After stained with phloroglucinol-HCl,the majority of the transformants overexpressing GbMYB1 were stained more intensely than the control plants.Compared with the control plants,the xylem cell walls of transgenic plants were thicker.These results indicate that GbMYB1 is involved in the regulation of the lignin biosynthetic pathway.In order to analyze the regulation of GbMYB1 transcription factor with genes in lignin biosynthetic pathway,a series of quantitative RT-PCR were carried out on the genes encoding lignin biosynthetic enzymes.The results showed that the expression of CADc,CADd and C3H genes in transgenic lines were up-regulated,especially the expression of CADc.Meanwhile,the gene PAL2 encoding the first primary enzyme in phenylpropanoid pathway was also up-regulated dramatically indicating that GbMYB1 transcription factor could up-regulate important genes involved in phenylpropanoid pathway and lignin biosynthetic pathway.On the other hand,Arabidopsis mutants AtMYB43,AtMYB98 showed opposite results to transgenic arabidopsis.The expressions of CADd and COMT genes encoding lignin biosynthetic enzyme were reduced obviously.All of these demonstrated directly and indirectly that GbMYB1 transcription factor is involved in phenylpropanoid pathway and lignin biosynthetic pathway.This study will be helpful for modification of phenylproanoid pathway and lignin biosynthetic pathway of G.biloba using metabolic engineering method in the near future.Another work in this study is the construction and screening of yeast one hybrid library from T.cuspidate.A yeast one hybrid library of T.cuspidate and five cis-elements involved in MeJA and SA induction were successfully constructed.And using GCC-box as bait,two putative transcription factors homeodomain TF and remorin TF were obtaind.更多还原