【作者】 王鹏；

【导师】 唐克轩；

【作者基本信息】 复旦大学 ， 遗传学， 2007， 博士

【摘要】 托品烷类生物碱是烟草中重要的一类次生代谢物。通过基因工程研究托品烷类生物碱的代谢调控模式,对于研究植物次生代谢物的调控、丰富代谢调控领域的知识具有重要的理论价值,并且对于降低烟草对人类健康的危害,提高烟草托品烷类生物碱的药用价值,以及提高烟草对害虫的抗性等方面具有潜在的应用价值。然而,这一领域在国内尚无人涉足,在国际上仅有的几篇报道中,因为样本数量、研究目标等的限制,研究结果提供的信息极为有限。本研究通过RNA介导的基因抑制,不仅使目标代谢物尼古丁的含量降低,而且通过扩大样本数量,获得了一系列的尼古丁含量降低的烟草,并在研究尼古丁合成主路与支路的关系的过程中,发现了新的代谢调控模式。本研究选取尼古丁合成的关键酶基因——腐胺甲基转移酶基因(PMT)作为调控靶点。此基因在烟草栽培种中是一个含有五条基因的小基因家族。选取其cDNA靠近3’端长540bp的保守序列作为基因抑制的目标序列,构建三种RNA介导的基因抑制载体——共抑制载体、反义抑制载体和RNA干扰载体,通过农杆菌介导的转化方法将其转入烟草叶盘,并通过筛选和鉴定,最终获得了645棵独立的转基因烟草植株,其中有217棵转基因植株获得了尼古丁含量数据。这些转基因烟草植株的表型与对照株相比,没有明显的表型上的异常。抑制效率最高的烟草植株由RNA干扰获得,其尼古丁含量仅为对照烟草植株中尼古丁含量的3.27%,而共抑制和反义抑制获得的抑制效率最高的烟草其尼古丁含量分别为对照植株的14.37%和7.91%。本研究获得了一系列的尼古丁含量降低程度的烟草:通过共抑制获得的烟草尼古丁含量降低了9.10%到85.63%,反义抑制烟草尼古丁含量降低了12.39%到92.09%,而RNA干扰烟草降低了29.00%到96.73%;并且每种方式获得的转基因烟草尼古丁含量从高到低均匀分布。实时定量PCR结果显示,尼古丁含量降低的转基因烟草植株PMT基因表达量相应降低;然而,尼古丁含量下降的程度没有PMT基因表达量降低的程度显著。尼古丁酸是尼古丁和安那他品生物合成的共同前体,它的合成受喹啉酸磷酸核糖转移酶控制。腐胺甲基转移酶基因抑制造成的尼古丁含量的降低必然导致吡啶环前体的累计,从而使安那他品含量上升。本研究证实了这个理论推测,并发现了一种新的代谢调控模式:PMT基因抑制后,随着尼古丁含量的逐步降低,安那他品含量并非呈线性提高,而是呈现台阶式的提高。在尼古丁含量降低程度不大时,安那他品的含量就会有所上升;但是,随着尼古丁含量的进一步降低,安那他品的含量并没有继续上升,而是维持在原来的升高的水平上;当尼古丁含量降低到3mg/g烟叶干重左右时,即尼古丁含量为对照烟草植株的10%左右时,安那他品的含量急剧上升,最高达到了2.1771mg/g烟叶干重。实时定量PCR结果表明,安那他品含量变化的这种模式,没有引起控制安那他品含量变化的关键酶基因QPT表达量的显著变化。更多还原

【Abstract】 Tropane alkaloids are important secondary metabolites in tobacco (Nicotiana tabacum).Studies on regulation modes of tropane alkaloids metabolism through genetic engineering,are academically important to enrich knowledge pool of metabolic regulation,and have potential value of application on reducing harm of cigarette on human health.These studies can also improve the pharmaceutical value of the tobacco tropane alkaloids in pharmaceutics,and enhance tobacco’ s resistance to insects. However,this field has yet to be studied in China,and only a few publications related to this field around the world provided limited messages due to restrictions such as amounts of samples and intentions of studying.Here tobacco lines with a series of reduced nicotine levels were obtained via RNA-mediated gene silencing and through extending amount of sample,and a new kind of regulation mode of tropane alkaloids metabolism was found related to nicotine biosynthesis and anatabine,a kind of product of its branched pathway.Genes encoding putrescine N-methyltransferase(PMT),the key enzyme controlling nicotine biosynthesis,were chosen to be targets to alter nicotine concentrations in tobacco.The gene family encoding this enzyme is constituted by five genes.Conserved sequence of 540bp which is near 3’ termini of PMT was selected to be target of silencing.Three kinds of RNA-mediated gene silencing vectors were constructed,that is,vector of cosuppression,that of antisense and that of RNA interference,which were transformed to tobacco leaf plates via Agrobacterium tumefaciens mediated transformation system.After screening and confirming,645 independent transgenic tobacco lines were generated,among which data of nicotine levels were obtained from 217 lines.The phenotype of the transgenic tobacco lines appears to be normal as compared to the control plants.RNAi approach was proved to be the most efficient way to reduce the levels of nicotine,whereas cosuppression and antisense approaches were less effective.The tobacco line of the most distinct silencing were obtained via RNA interference,whose nicotine level is 3.27%compared to CK line. Nicotine level of the tobacco line of the most distince silencing obtained via cosuppression and antisense is 14.37%and 7.91%compared to CK line respectively.As to cosuppression lines,nicotine contents were reduced between 9.10%and 85.63%;as to antisense lines,between 12.39%and 92.09% RNAi lines,between 29.00%and 96.73%.Nicotine levels of these lines distributed averagely.Results of RT-QPCR showed that there is positive correlation between nicotine contents and expression of PMT,and reduction of nicotine is not as evident as depression of PMT expression.Nicotinic acid is the precursor of both anatabine and the pyridine ring of nicotine,whose synthesis is regulated by quinolinate phosphoribosyltransferase(QPT).Down regulation of PMT activity in Nicotiana would be expected to lead to elevated levels of anatabine along with reduction of nicotine levels.In this study,tobacco lines with silenced PMT showed an intriguing mode for variety of anatabine levels. Reduction of the level of nicotine does not result in elevation of anatabine levels until nicotine concentration were lower than 3mg/g, which is about 10%compared of that of CK,when a dramatically elevated anatabine level were observed.Anatabine level of the most notable line was 2.1771mg/g.Results of RT-QPCR showed no distinct change of QPT along with elevation of anatabine levels.更多还原