【作者】 杨青杰；

【导师】 朱长甫；

【作者基本信息】 东北师范大学 ， 植物学， 2013， 博士

【摘要】 类胡萝卜素是自然界的一大类萜类化合物，在动物营养及花色研究中占有重要地位。近20年来，类胡萝卜素基因克隆和功能鉴定及遗传工程研究工作取得了很大进展，尤其在农作物的类胡萝卜素含量提高方面取得了显著的成效，但对于植物类胡萝卜素代谢过程相关调控机制的研究还相对不足。因此，在应用遗传工程手段对作物进行遗传育种操作时存在很大的盲目性[2]。为了使类胡萝卜素基因工程研究更加有的放矢，当前更需要对植物体内的类胡萝卜素合成相关基因的表达调控机制进行深入研究。启动子在调控基因表达过程中起关键作用，因此启动子及其顺式作用元件的生物学功能研究是探求基因表达调控机制的重要部分。类胡萝卜基因的转录控制着黄龙胆花发育期间类胡萝卜素的积累。本研究通过分离并分析黄龙胆类胡萝卜素生物合成途径中相关酶基因的启动子来研究黄龙胆花发育期间类胡萝卜素基因表达协同上调的分子调控机制，试图在克隆到的协同上调的类胡萝卜素基因启动子调控区域找到共有顺式调控元件。研究成果如下：（一）本研究中共克隆得到6个与类胡萝卜素生物合成相关的启动子，即黄龙胆八氢番茄红素脱氢酶(GlZEP)基因启动子、黄龙胆八氢番茄红素脱氢酶(GlPDS)基因启动子、ζ-胡萝卜素脱氢酶(GlZDS)基因启动子、番茄红素β-环化酶(GlLYCB)基因启动子、胡萝卜素β-环羟化酶(GlBCH）基因启动子和番茄红素ε-环化酶(GlLYCE)基因启动子，启动子全长分别为2225bp、1077bp、766bp、1506bp、1744bp和938bp。（二）本研究中对各启动子的顺式作用元件分析得出如下结论：1、 GlZEP基因启动子序列-72bp和-84bp处有2个TATA-box；有6个对真核生物起到增强基因转录效率的CAAT-box；有较多的光调控顺式作用元件，包括：1个GT1元件、2个box I、3个G box、1个GAG元件、4个box4和1个chs-CMA2a元件；还发现几个与激素或逆境响应有关的顺式作用元件，包括1个乙烯响应元件ERE、2个CGTCA元件（对茉莉酸甲酯敏感）、2个涉及干旱胁迫的MYB结合位点、1个热激元件、3个真菌诱导响应的Box-W1和一个昼夜节律控制元件。2、 GlZEP、GlPDS、GlZDS、GlLYCB、GlBCH和GlLYCE基因启动子除核心启动子元件外，所有的启动子均包括三个顺式调控元件的共有元件：1）涉及MeJA-响应的CGTCA-元件；2)最近发现的在拟南芥PSY启动子上与RAP2.2有相互作用的ATCTA-元件；3）胚乳表达所必需的Skn-1元件。3、 GlZEP、GlPDS、GlZDS、GlLYCB、GlBCH和GlLYCE基因启动子序列中都发现至少一种类型的光响应元件，这些启动子可能根据日长和其他涉及花发育调控事件来进行类胡萝卜素表达调控。4、 GlZEP、GlPDS、GlZDS、GlLYCB、GlBCH和GlLYCE基因启动子序列中发现至少一种类型的MBS元件(涉及干旱诱导的MYB结合位点)。5、在GlPDS和GlLYCE基因启动子中存在乙烯响应元件ERE；GlLYCE和GlBCH基因启动子中存在ABRE（涉及脱落酸应答的顺式作用元件）和生长素响应元件（TGA-盒或TGA-元件)； GlLYCB和GlLYCE基因启动子中存在对真菌感染起应答功能的Box-W1；GlZDS, GlLYCB和GlLYCE基因启动子中存在GCN4（涉及胚乳表达的顺式作用元件）；GlLYCE和GlBCH基因启动子中还分别检测到了富TA区（烟草中的转录增强子）和ATGCAA-元件（水稻中与TGAGTCA元件(GCN4)相联系的结合位点）。（三）本研究中通过各启动子片段在番茄（Solanum lycopersicum cv. Micro-Tom）果实中的瞬时表达及GlZEP基因启动子在转基因番茄中的稳定表达，得出如下结论：1、对GlZEP基因启动子的全长及缺失片段的研究显示，GlZEP基因启动子下游的5’非编码区所包含的序列对于所驱动基因的高水平表达是必要的。GlZEP基因启动子近端的677bp区域内包含了其保持基本活性所必需的所有顺式作用元件。2、在含有有色体的成熟绿色果实(MG)中瞬时表达PDS-gusA、ZDS-gusA、LYCB-gusA、BCH-gusA和LYCE-gusA，所有启动子都能驱动转基因番茄中gusA报告基因的发育调控表达，表达模式与GLZEP基因启动子相似。但在未成熟绿色果实(IM)中这些启动子驱动的gusA基因则呈现非常低的表达水平或几乎不表达。更多还原

【Abstract】 Carotenoids are naturally tetraterpenoids. They are important in nutuition for human. Inaddition carotenoids function as attractants to pollinators and seed dispersal agents. Duringthe last two decades, significant advances have been made in cloning most of thecarotenogenic genes and the genetic engineering of plants with the intention of manipulationof carotenoid content. However, a major limitation is our poor knowledge of how endogenouscarotenogenic genes are regulated in higher plants. Poor knowledge of the regulation ofcarotenogenic gene expression has thus far limited the predictive carotenoid engineering ofplants for enhancing carotenoid content and composition.The investigation of promoter was one of means for attempts to overcome theseroadblocks in plants by breaking through them or going around them. So the promoter andtheir cis-elements were researched. During the development of Gentiana lutea petals, atemporal correlation among the accumulation of carotenoids, the formation of chromoplasts,and the coordinated up-regulation of carotenogenic gene expression is evident. In this report,in order to elucidate the molecular control mechanisms of the coordinated up-regulation ofcarotenogenic gene expression, six promoters (GlZEP, GlPDS, GlZDS, GlLYCB, GlBCH andGlLYCE) were isolated by inverse PCR from G. lutea. All of these promoters were able todrive developmentally regulated expression of the gusA reporter gene in transgenic tomato(Solanum lycopersicum cv. Micro-Tom).We transformed tomato plants with the gusA gene encoding the reporter enzymeβ-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated thereporter expression profile. We detected high levels of gusA expression and GUS activity inchromoplast containing flowers and fruits, but minimal levels in immature fruits containinggreen chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictlyassociated with fruit development and chromoplast differentiation, suggesting anevolutionarily-conserved link between ZEP and the differentiation of organelles that storecarotenoid pigments.We transformed tomato plants with the gusA gene encoding the reporter enzymeβ-glucuronidase (GUS) under the control of the five genes(GlPDS, GlZDS, GlLYCB, GlBCHand GlLYCE) promoters respectively. High levels of gusA expression driven by thesepromoters were found in chromoplast-containing mature green (MG) fruits, but very lowlevels of expression, if any, were seen in chloroplast-containing immature green (IM) fruitsfor both GlLYCB and GlBCH promoters. The expression of these promoters is strictlyassociated with tomato fruit development and chromoplast differentiation. In addition to corepromoter elements (TATA box and CAAT box), three common cis-regulatory motifs involvedin methyl jasmonate (MeJA) responsiveness (CGTCA-motif), and binding an ethyleneresponse transcription factor (ATCTA-motif)，required for endosperm expression Skn-1motif(GTCAT) were found in all the carotenogenic gene (GlPDS, GlZDS, GlLYCB, GlBCH, GlZEP and GlLYCE) promoters of G. lutea. Our present investigation provides new insights into theregulatory architecture of the coordinated up-regulation of carotenogenic gene expression inpetals of G. lutea. The impact of our results on current models for the regulation ofcarotenogenesis in plants is discussed.更多还原