中国水仙DFR基因启动子的克隆及功能

为了解中国水仙花青素合成途径关键基因DFR(NtDFR)的表达调控以及中国水仙不能合成花青素的分子机制,采用染色体步移法从中国水仙中克隆NtDFR基因起始密码子上游962 bp的启动子序列.生物信息学分析结果表明启动子序列除包含TATA-box、CAAT-box等基本启动子元件外,还包含光调控元件、植物激素响应元件、胁迫响应元件等多个顺式作用元件.此外,该启动子序列还含有MYB转录因子结合位点.为验证启动子的表达特性,将NtDFR启动子取代植物表达载体pBI121上35S启动子,构建pBI121-pNtDFR::GUS载体,利用农杆菌转化烟草叶片瞬时表达,通过GUS组织染色法确定了克隆的启动子的活性.将中国水仙R2R3-MYB转录因子NtMYB2、NtMYB5分别和pBI121-p NtDFR::GUS共同注射烟草,定量PCR和GUS组织化学染色结果表明NtMYB2和NtMYB5都使NtDFR启动子诱导的烟草叶片GUS颜色变浅以及GUS基因表达量下降,表明NtMYB2和NtMYB5是NtDFR的抑制因子.本研究结果有助于了解中国水仙花青素合成途径的分子调控机制.(图5参34)     

青稞β-1,3-葡聚糖酶II基因启动子的克隆分析及表达载体的构建

根据本实验室克隆并公布的藏青稞β-1,3-葡聚糖酶II(BGH II)基因序列,以藏青稞QB09基因组DNA为模板,构建DNA步移文库,并通过步移技术克隆出BGH II起始密码子上游调控序列BGHP. 鉴定和分析表明,BGHP具备大多数高等植物启动子的保守元件,预测它对BGH II基因的表达具有一定的作用.为鉴定BGH II基因的基本启动子元件,将基因5′侧翼序列做缺失片段分析,利用PCR方法从BGHP中得到3个不同大小两端带有Hind Ⅲ、BamHⅠ酶切位点的片段BGHP1、BGHP2和BGHP3,定向插入载体pMGFP4(pBI221改建,报告基因为GFP)中,取代原有的CaMV35S启动子,构建了由驱动报告基因GFP的植物表达载体BGHPV1、BGHPV2和BGHPV3,用于大麦的遗传转化,为进一步研究其功能奠定了基础. 图5 表1 参15     

青稞β-1,3-葡聚糖酶Ⅱ基因启动子的克隆分析及表达载体的构建

根据本实验室克隆并公布的藏青稞β-1,3-葡聚糖酶Ⅱ(BGH Ⅱ)基因序列,以藏青稞QB09基因组DNA为模板,构建DNA步移文库,并通过步移技术克隆出BGH Ⅱ起始密码子上游调控序列BGHP.鉴定和分析表明,BGHP具备大多数高等植物启动子的保守元件,预测它对BGHⅡ基因的表达具有一定的作用.为鉴定BGHⅡ基因的基本启动子元件,将基因5’侧翼序列做缺失片段分析,利用PCR方法从BGHP中得到3个不同大小两端带有HindⅢ、BamH Ⅰ酶切位点的片段BGHP1、BGHP2和BGHP3,定向插入载体pMGFP4(pBI221改建,报告基因为GFP)中,取代原有的CaMV35S启动子,构建了由驱动报告基因GFP的植物表达载体BGHPV1、BGHPV2和BGHPV3,用于大麦的遗传转化,为进一步研究其功能奠定了基础.图5表1参15     

青稞β—1，3—葡聚糖酶Ⅱ基因启动子的克隆分析及表达载体的构建

根据本实验室克隆并公布的藏青稞β—1，3—葡聚糖酶Ⅱ(BGH Ⅱ)基因序列，以藏青稞QB09基因组DNA为模板，构建DNA步移文库，并通过步移技术克隆出BGH Ⅱ起始密码子上游调控序列BGHP．鉴定和分析表明，BGHP具备大多数高等植物启动子的保守元件，预测它对BGHⅡ基因的表达具有一定的作用．为鉴定BGHⅡ基因的基本启动子元件，将基因5′侧翼序列做缺失片段分析，利用PCR方法从BGHP中得到3个不同大小两端带有Hind Ⅲ、BamH I酶切位点的片段BGHP1、BGHP2和BGHP3，定向插人载体pMGFP4(pBI221改建，报告基因为GFP)中，取代原有的CaMV35S启动子，构建了由驱动报告基因GFP的植物表达载体BGHPVl、BGHPV2和BGHPV3，用于大麦的遗传转化，为进一步研究其功能奠定了基础．图5表1参15     

水稻花药特异表达启动子的克隆与活性检测

植物雄性器官特异表达启动子的克隆是作物杂种优势利用分子育种的基础.根据水稻花药特异表达RA8基因序列设计引物,从水稻(Oryza sativa L.)品种日本晴中克隆得到水稻花药特异表达基因RA8启动子Tsp2,该启动子为RA8基因翻译起始位点上游828 bp的片段,具有启动子特征序列TATA盒TATAAATA和真核生物启动子特征序列CAAT框,与RA8启动子的核苷酸序列同源性为97%.利用该启动子与报告基因GFP构建植物表达载体p1304-rap,采用基因枪法转化烟草花药,通过GFP的瞬时表达证明该启动子具有花药特异启动子活性. 图5 参9     

一个硫化叶菌病毒启动子的分离与鉴定

几乎所有古菌病毒基因组中无RNA聚合酶(RNA polymerase,RNAP)等组成基本转录装置的同源蛋白编码序列,而且启动子活性对病毒感染过程中病毒基因的转录上可能具有重要的影响.为进一步揭示古菌病毒基因启动子的序列结构特点和活性之间的关系,首先基于硫化叶菌质粒pSeSD,将β-半乳糖苷酶编码基因lacS克隆到阿拉伯糖启动子araS下游多克隆位点,构建重组表达载体pSeSD-lacS.将pSeSD-lacS转化冰岛硫化叶菌(Sulfolobus islandicus)E233S菌株后的功能分析结果表明,lacS基因成功表达.在此基础上,利用硫化叶菌病毒STSV2衣壳蛋白编码基因ORF37上游500bp的潜在启动子片段P37替换pSeSD-lacS中的araS启动子,构建出新的重组表达质粒pSeSD-P37-lacS,进一步将pSeSD-P37-lacS转化E233S菌株进行启动子活性分析.β-半乳糖苷酶酶活结果显示,诱导后araS启动子酶活为14 345.7±422.3 mU,P37酶活为13 723.1±370.9 mU,表明P37片段具有启动子功能,而且活性与araS启动子相当.序列分析也显示,P37具有与硫化叶菌基因启动子类似的基础序列元件initiator、TATA-box及BRE等.本研究表明pSeSD-lacS可作为一个硫化叶菌病毒基因启动子筛选载体,而且高活性的基因启动子可能在STSV2病毒生命过程具有重要的作用.(图4表1参27)     

番茄HP1和HP2基因RNA共干涉载体的构建及遗传转化

番茄HP1和HP2是色素积累的负调控因子,在光形态建成和色素积累调控中起着重要作用.将番茄HP1、HP2基因片段导入到植物表达载体pBI121,用番茄果实特异表达的TMF7基因的启动子替换原有的CaMV 35S启动子,构建果实特异表达HP1、HP2双基因RNA共干涉植物表达载体pBI121-TMF7-HP1HP2.通过根癌农杆菌介导转入番茄子叶,经组织培养成功获得转基因植株.半定量RT-PCR分析显示,转基因植株果实内HP1、HP2的表达量均明显低于野生型植株果实.转基因植株果实叶绿素含量比野生型明显升高,而叶片中的叶绿素含量无明显差异.该研究结果为采用基因工程的方法改善番茄果实营养品质作出了新的尝试和提出了新的思路.图6表2参15     

人类无精症相关新基因ZNF313启动子的初步分析

采用PCR方法扩增了ZNF313基因的启动子序列,构建了含人ZNF313基因启动子不同片断的荧光素酶报告基因表达体系.以pRLTK为内参照质粒,瞬时转染HEK293T细胞,48h后收集细胞,测定荧光素酶的相对表达活性.结果发现,在ZNF313基因的启动子区域构建了4种荧光素酶报告基因表达体系,即pGL3215(-215bp～ 38bp)、pGL3160(-160bp～ 38bp)、pGL3133(-133bp～ 128bp)和pGL38(-8bp～ 128bp).其中pGL3215表达载体的荧光素酶相对表达活性最高;pGL3160和pGL3133表达载体的荧光素酶相对表达活性几乎相同,且是pGL3215的75%;而pGL38的荧光素酶相对表达活性急剧下降,接近于零.这表明,-133bp～-8bp区域内含有人ZNF313基因转录所必需的启动子序列.生物信息学的分析表明,两个SP1、一个AP2和一个TAg是人ZNF313基因启动子所必需的.图4参19     

番茄ARF4基因果实特异RNAi载体的构建及遗传转化

构建ARF4基因果实特异表达的RNA干涉载体,对转基因番茄果实进行初步分析,可为采用基因工程方法改良番茄果实品质做出新尝试.利用RT-PCR技术从番茄果实cDNA中扩增SlARF4基因全长,将番茄ARF4基因正反向重复序列片段导入到植物表达载体pBI121上,启动子是番茄果实特异表达的TFM7.将构建的ARF4基因果实特异RNA干涉载体pBI121-TFM7-A4Ri通过根癌农杆菌介导转入到野生型番茄中,进而对转化获得的植株进行了阳性鉴定.分别以转基因番茄和野生型番茄为材料,分析ARF4在果实中的表达水平,测定绿熟期果实叶绿素含量、果实的单果重量和果皮厚度.酶切证实pBI121-TFM7-A4Ri果实特异表达载体构建成功,而且,PCR检测也得到阳性转基因株.半定量RT-PCR分析显示,转基因番茄果实中ARF4的表达量明显低于野生型果实.转基因番茄果实的叶绿素含量、单果重量和果皮厚度都比野生型有提高.因此,ARF4果实特异表达的RNAi方法能够改良番茄果实品质.     

Valuation of human health: An integrated model of willingness to pay for mortality and morbidity risk reductions

This paper develops and applies an integrated model of mortality and morbidity valuation that is consistent with the principles of welfare economics. To obtain the integrated model, the standard one-period expected utility model of one person facing the prospect of either being alive or dead is extended to incorporate (1) a third health state (sick) with a utility level that is intermediate to utility if healthy and utility if dead, (2) a family perspective in which a parent makes choices about risk exposure both for herself and for a child, and (3) a multi-period framework that allows for possible parent/child differences in illness latency. Monetary benefits of health risk reduction obtained from the integrated model are compared with those that would be computed using the standard model. The integrated model then is applied using data obtained from two field studies of skin cancer and leukemia to demonstrate how it can be used to estimate health benefits of reduced illness and death risks.     

Price controls and banking in emissions trading: An experimental evaluation

We present results from laboratory emissions markets designed to investigate the effects of price controls and permit banking on limiting permit price risk. While both instruments reduce between-period price volatility and within-period price dispersion, combining price controls and permit banking yields important benefits. Banking alone produces high permit prices in earlier periods that fall over time, but the combined policy produces lower initial prices and lower volatility. However, banking, price controls, and the combination all produce higher between-period emissions volatility. Hence, for emissions markets that seek to control flow pollutants with strictly convex damages, efforts to limit permit price risk can result in higher expected damage.     

Government green procurement spillovers: Evidence from municipal building policies in California

We study how government green procurement policies influence private-sector demand for similar products. Specifically, we measure the impact of municipal policies requiring governments to construct green buildings on private-sector adoption of the US Green Building Council?s Leadership in Energy and Environmental Design (LEED) standard. Using matching methods, panel data, and instrumental variables, we find that government procurement rules produce spillover effects that stimulate both private-sector adoption of the LEED standard and investments in green building expertise by local suppliers. These findings suggest that government procurement policies can accelerate the diffusion of new environmental standards that require coordinated complementary investments by various types of private adopter.     

Innovation and the dynamics of global warming

Global warming and the carbon cycle are a dynamic system with positive feedbacks. Fossil fuels are exhaustible resources. These two facts mean that innovation in clean energy technology, rather than mitigating global warming, can lead to a permanently higher temperature path. This paper explores the impact of innovation in the simplest model linking the economic theory of exhaustible resources with positive feedback dynamics in the carbon cycle.     

Market equilibrium and welfare effects of a fuel tax in China: The impact of consumers' response through driving patterns

We investigate the market equilibrium and welfare effects of a fuel tax in China relative to an alternative policy instrument that rations the number of new automobile sales through auctioned quotas. Unlike those of previous studies, our modeling approach incorporates both household car purchase and utilization decisions, the latter of which have been ignored in previous studies on China's fuel tax. Ignoring this margin of choice will underestimate the fuel tax's ability to mitigate externalities. Using detailed household-level panel data and a fixed effects econometric specification, we estimate the fuel price elasticity of vehicle miles traveled is −0.59 on average. The results of the counterfactual analysis suggest that a 51% increase in tax-inclusive gasoline prices will reduce car sales by 24.9% but increase social welfare to a degree that depends on vehicles' lifetime. We find that compared to auctioned quotas, the fuel tax results in greater car sales but higher social welfare.     

Good news for environmental self-regulation? Finding the right link

We investigate the stock market response to firm disclosure of positive environmental information and the link from that information to environmental outcomes. We classify environmental media releases by informational content and value relevance, and assess the abnormal stock returns of each type of event. While announcements of future environmental activities lead to the largest favorable stock market reactions, there is no guaranteed link from this type of information to environmental outcomes. Further analysis of the abnormal returns shows that the magnitude of the stock market reaction depends on firm financial characteristics across all event types rather than on firm environmental performance. Our results indicate that the ability for voluntary environmental information disclosure to induce environmental self-regulation is limited to the extent that firms are able to follow through with their announcements of planned environmental activities.     

Prospect theory and energy efficiency

Investments in energy efficiency entail uncertainty, and when faced with uncertainty consumers have been shown to behave according to prospect theory: preferences are reference-dependent and exhibit loss aversion, and probabilities are subjectively weighted. Using data from a choice experiment eliciting prospect theory parameters, I provide evidence that loss-averse people are less likely to invest in energy efficiency. Then, I consider policy design under prospect theory when there are also externalities from energy use. A higher degree of loss aversion implies a higher subsidy to energy efficiency. Numerical simulations suggest that the impact of prospect theory on policy may be substantial.     

Strategic pollution control and capital tax competition

In an analytical model of symmetric countries with mobile capital and local or transboundary pollution we investigate whether competition in emissions taxes (or emissions caps) and capital taxes leads to efficient outcomes when governments act strategically. When they have capital taxes and emissions caps at their disposal, they refrain from taxing capital and set their caps inefficiently lax [efficient] for transboundary [local] pollution. When they have the option to tax capital and emissions, capital is subsidized [untaxed] and emissions taxes are inefficiently low [efficient] for transboundary [local] pollution. In case of transboundary pollution emissions caps are Pareto-superior to emissions taxes. That holds regardless of whether the environmental policy is applied as stand-alone policy or combined with capital tax competition.     

The Antiquities Act,national monuments,and the regional economy

Large, landscape-scale national monuments have long been controversial. It has been claimed that large monuments harm local economies by restricting growth of the grazing, timber, mining, and energy industries. Others have asserted that large monuments aid economic growth by reducing reliance on volatile commodity markets and fostering tourism growth. In this study, we use a synthetic control approach to measure the average causal effect of nine national monument designations on county-level per capita income. We find no evidence that monument designation affected per capita income in any of 20 counties hosting nine large (>50,000 acres) national monuments established under the Antiquities Act (six monuments) or by legislative action (three monuments). The broad economic claims of both advocates and critics of large national monuments have little empirical support. The absence of a designation effect for large national monuments is likely due to the attributes of federal land and the legal constraints under which it is managed.     

Environmental policy and human capital inequality: A matter of life and death

This paper analyzes the economic implications of an environmental policy when we account for the life expectancy of heterogeneous agents. In a framework in which everyone suffers from pollution but health status also depends on individual human capital, we find that the economy may be stuck in a trap in which inequality rises steadily, especially when the initial pollution intensity of production is too high. We emphasize that such inequality is in the long run costly for the economy in terms of health and growth. Therefore, we study whether a tax on pollution associated with an investment in pollution abatement can be used to address this situation. We show that a stricter environmental policy may allow the economy to escape from the inequality trap while enhancing the long-term growth rate when the initial inequality in human capital is not too large.