三个方便实用的植物表达载体构建与验证

为满足植物功能基因组学研究及转基因安全性需要,本研究根据一些国内外引进或商业化的植物表达载体及其相关元件,构建了3个适合于植物,尤其是单子叶植物转化的表达载体,即p AH006、p WMB022和p WMB025。p AH006载体包含由玉米泛素ubi启动子调控的GUS基因和bar基因的完整T-DNA区域,此区段能够被酶切回收,可用于单子叶植物农杆菌介导转化效率评价及基因枪介导线状DNA转化效果研究;p WMB022载体携带由双35S启动子调控的玉米色素基因Lc和C1,可用作基因枪介导的共转化筛选标记,直观筛选含目标基因转基因材料;p WMB025载体携带由ubi启动子调控的、商业化转基因植物中广泛利用的EPSPS基因,可用于禾谷类作物农杆菌或基因枪介导的遗传转化,载体多克隆位点可通过酶切方式更换目标基因。酶切鉴定结合农杆菌或基因枪介导的小麦幼胚愈伤组织或叶片转化验证此3个载体表明,载体构建正确,其标记基因、可视化基因和报告基因均能正常表达。这3个载体的构建对于小麦等植物转化效率提升、安全型转基因作物获得和植物功能基因组学研究等具有重要意义。     

Impact of agronomic uncertainty in biomass production and endogenous commodity prices on cellulosic biofuel feedstock composition

This study evaluates the effect of agronomic uncertainty on bioenergy crop production as well as endogenous commodity and biomass prices on the feedstock composition of cellulosic biofuels under a binding mandate in the United States. The county‐level simulation model focuses on both field crops (corn, soybean, and wheat) and biomass feedstocks (corn stover, wheat straw, switchgrass, and Miscanthus). In addition, pasture serves as a potential area for bioenergy crop production. The economic model is calibrated to 2022 in terms of yield, crop demand, and baseline prices and allocates land optimally among the alternative crops given the binding cellulosic biofuel mandate. The simulation scenarios differ in terms of bioenergy crop type (switchgrass and Miscanthus) and yield, biomass production inputs, and pasture availability. The cellulosic biofuel mandates range from 15 to 60 billion L. The results indicate that the 15 and 30 billion L mandates in the high production input scenarios for switchgrass and Miscanthus are covered entirely by agricultural residues. With the exception of the low production input for Miscanthus scenario, the share of agricultural residues is always over 50% for all other scenarios including the 60 billion L mandate. The largest proportion of agricultural land dedicated to either switchgrass or Miscanthus is found in the southern Plains and the southeast. Almost no bioenergy crops are grown in the Midwest across all scenarios. Changes in the prices for the three commodities are negligible for cellulosic ethanol mandates because most of the mandate is met with agricultural residues. The lessons learned are that (1) the share of agricultural residue in the feedstock mix is higher than previously estimated and (2) for a given mandate, the feedstock composition is relatively stable with the exception of one scenario.     

The Feasibility of Producing Adequate Feedstock for Year-Round Cellulosic Ethanol Production in an Intensive Agricultural Fuelshed

To date, cellulosic ethanol production has not been commercialized in the United States. However, government mandates aimed at increasing second-generation biofuel production could spur exploratory development in the cellulosic ethanol industry. We conducted an in-depth analysis of the fuelshed surrounding a starch-based ethanol plant near York, Nebraska that has the potential for cellulosic ethanol production. To assess the feasibility of supplying adequate biomass for year-round cellulosic ethanol production from residual maize (Zea mays) stover and bioenergy switchgrass (Panicum virgatum) within a 40-km road network service area of the existing ethanol plant, we identified ～14,000 ha of marginally productive cropland within the service area suitable for conversion from annual rowcrops to switchgrass and ～132,000 ha of maize-enrolled cropland from which maize stover could be collected. Annual maize stover and switchgrass biomass supplies within the 40-km service area could range between 429,000 and 752,000 metric tons (mT). Approximately 140–250 million liters (l) of cellulosic ethanol could be produced, rivaling the current 208 million l annual starch-based ethanol production capacity of the plant. We conclude that sufficient quantities of biomass could be produced from maize stover and switchgrass near the plant to support year-round cellulosic ethanol production at current feedstock yields, sustainable removal rates and bioconversion efficiencies. Modifying existing starch-based ethanol plants in intensive agricultural fuelsheds could increase ethanol output, return marginally productive cropland to perennial vegetation, and remove maize stover from productive cropland to meet feedstock demand.     

Downregulation of Cinnamyl Alcohol Dehydrogenase (CAD) Leads to Improved Saccharification Efficiency in Switchgrass

The bioconversion of carbohydrates in the herbaceous bioenergy crop, switchgrass (Panicum virgatum L.), is limited by the associated lignins in the biomass. The cinnamyl alcohol dehydrogenase (CAD) gene encodes a key enzyme which catalyzes the last step of lignin monomer biosynthesis. Transgenic switchgrass plants were produced with a CAD RNAi gene construct under the control of the maize ubiquitin promoter. The transgenic lines showed reduced CAD expression levels, reduced enzyme activities, reduced lignin content, and altered lignin composition. The modification of lignin biosynthesis resulted in improved sugar release and forage digestibility. Significant increases of saccharification efficiency were obtained in most of the transgenic lines with or without acid pretreatment. A negative correlation between lignin content and sugar release was found among these transgenic switchgrass lines. The transgenic materials have the potential to allow for improved efficiency of cellulosic ethanol production.     

A set of modular binary vectors for transformation of cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.     

Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production

Switchgrass (Panicum virgatum L.) has been developed into a dedicated herbaceous bioenergy crop. Biomass yield is a major target trait for genetic improvement of switchgrass. microRNAs have emerged as a prominent class of gene regulatory factors that has the potential to improve complex traits such as biomass yield. A miR156b precursor was overexpressed in switchgrass. The effects of miR156 overexpression on SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were revealed by microarray and quantitative RT-PCR analyses. Morphological alterations, biomass yield, saccharification efficiency and forage digestibility of the transgenic plants were characterized. miR156 controls apical dominance and floral transition in switchgrass by suppressing its target SPL genes. Relatively low levels of miR156 overexpression were sufficient to increase biomass yield while producing plants with normal flowering time. Moderate levels of miR156 led to improved biomass but the plants were non-flowering. These two groups of plants produced 58%-101% more biomass yield compared with the control. However, high miR156 levels resulted in severely stunted growth. The degree of morphological alterations of the transgenic switchgrass depends on miR156 level. Compared with floral transition, a lower miR156 level is required to disrupt apical dominance. The improvement in biomass yield was mainly because of the increase in tiller number. Targeted overexpression of miR156 also improved solubilized sugar yield and forage digestibility, and offered an effective approach for transgene containment.     

Bioenergy crop productivity and potential climate change mitigation from marginal lands in the United States: An ecosystem modeling perspective

Growing biomass feedstocks from marginal lands is becoming an increasingly attractive choice for producing biofuel as an alternative energy to fossil fuels. Here, we used a biogeochemical model at ecosystem scale to estimate crop productivity and greenhouse gas (GHG) emissions from bioenergy crops grown on marginal lands in the United States. Two broadly tested cellulosic crops, switchgrass, and Miscanthus, were assumed to be grown on the abandoned land and mixed crop‐vegetation land with marginal productivity. Production of biomass and biofuel as well as net carbon exchange and nitrous oxide emissions were estimated in a spatially explicit manner. We found that, cellulosic crops, especially Miscanthus could produce a considerable amount of biomass, and the effective ethanol yield is high on these marginal lands. For every hectare of marginal land, switchgrass and Miscanthus could produce 1.0–2.3 kl and 2.9–6.9 kl ethanol, respectively, depending on nitrogen fertilization rate and biofuel conversion efficiency. Nationally, both crop systems act as net GHG sources. Switchgrass has high global warming intensity (100–390 g CO₂eq l^?1 ethanol), in terms of GHG emissions per unit ethanol produced. Miscanthus, however, emits only 21–36 g CO₂eq to produce every liter of ethanol. To reach the mandated cellulosic ethanol target in the United States, growing Miscanthus on the marginal lands could potentially save land and reduce GHG emissions in comparison to growing switchgrass. However, the ecosystem modeling is still limited by data availability and model deficiencies, further efforts should be made to classify crop‐specific marginal land availability, improve model structure, and better integrate ecosystem modeling into life cycle assessment.     

Environmental and economic analysis of the fully integrated biorefinery

Cellulosic biofuel systems have the potential to significantly reduce the environmental impact of the world's transportation energy requirements. However, realizing this potential will require systems level thinking and scale integration. Until now, we have lacked modeling tools for studying the behavior of integrated cellulosic biofuel systems. In this paper, we describe a new research tool, the Biorefinery and Farm Integration Tool (BFIT) in which the production of fuel ethanol from cellulosic biomass is integrated with crop and animal (agricultural) production models. Uniting these three subsystems in a single combined model has allowed, for the first time, basic environmental and economic analysis of biomass production, possible secondary products, fertilizer production, and bioenergy production across various regions of the United States. Using BFIT, we simulate cellulosic ethanol production embedded in realistic agricultural landscapes in nine locations under a collection of farm management scenarios. This combined modeling approach permits analysis of economic profitability and highlights key areas for environmental improvement. These results show the advantages of introducing integrated biorefinery systems within agricultural landscapes. This is particularly true in the Midwest, which our results suggest is a good setting for the cellulosic ethanol industry. Specifically, results show that inclusion of cellulosic biofuel systems into existing agriculture enhances farm economics and reduces total landscape emissions. Model results also indicate a limited ethanol price effect from increased biomass transportation distance. Sensitivity analysis using BFIT revealed those variables having the strongest effects on the overall system performance, namely: biorefinery size, switchgrass yield, and biomass farm gate price.     

Bioethanol from maize cell walls: genes,molecular tools,and breeding prospects

In the last decade, cellulosic ethanol has caught the growing interest of governments and private investors worldwide as it brings the promise of responsible renewable-energy and an opportunity to depart from an oil-reliant economy. Alongside advances in bioprocessing technologies, the development of specialized bioenergy crops is seen as a pressing industrial necessity, and while C4 perennials (e.g., Miscanthus, switchgrass, and sugarcane) have been coined the most promising candidates for the production of lignocellulosic biomass, maize should not be overlooked. In this review, we have addressed the benefits of advancing maize as a second-generation bioenergy feedstock. We have also analyzed current knowledge on the maize cell wall and promising genetic strategies for its modification, given that lignocellulose recalcitrance represents the most crucial breeding target in bioenergy crop research programs. In addition to lignin, a focus on the underlying genetic basis of cellulose, hemicellulose, and ferulate cross-linking patterns, as well as their regulation, has been warranted. A comprehensive overview of the state-of-art of genomic and phenotyping strategies available for bioenergy crop research is also provided. Overall, maize represents an outstanding model organism for understanding complex cell wall characteristics and defining the path for breeders looking to improve this and other promising bioenergy grasses. With an extensive array of dedicated agronomic and genomic resources at hand, we believe that breeding maize with improved processing amenability is a likely prospect but would like to remind readers that advances in high-biomass yielding properties, improved agronomic hardiness, and enhanced processing efficiency will also be necessary.     

Ammonia fiber expansion (AFEX) treatment of eleven different forages: Improvements to fiber digestibility in vitro

With the increased attention to bioenergy and especially cellulosic ethanol, there are concerns regarding potential competition for available land between biofuels and feeds/foods. Ammonia fiber expansion (AFEX), a pretreatment process for cellulosic ethanol, may also be used to improve ruminant digestibility of feedstuffs not traditionally used as forages. Eleven forages – including traditional forages, agricultural residues, and dedicated energy crops – were AFEX treated and digested in vitro with rumen inoculum. AFEX treatment improved 48-h neutral detergent fiber (aNDFom) digestion for several moderately indigestible forages compared to untreated samples, but showed no improvement for highly digestible samples. Of particular interest are corn stover and late-harvest switchgrass, as AFEX treatment improved digestibility by 52% and 128% over untreated material, whereas the improvement was 74% and 70% over conventional ammonia treatment, respectively. The crude protein content of all treated samples increased to more than 100 g/kg dry forage. This research strongly suggests that AFEX-treated feedstuffs can be competitive with traditional forages, and thus offer expanded options for ruminant feeding.     

Mapping marginal croplands suitable for cellulosic feedstock crops in the Great Plains,United States

Growing cellulosic feedstock crops (e.g., switchgrass) for biofuel is more environmentally sustainable than corn‐based ethanol. Specifically, this practice can reduce soil erosion and water quality impairment from pesticides and fertilizer, improve ecosystem services and sustainability (e.g., serve as carbon sinks), and minimize impacts on global food supplies. The main goal of this study was to identify high‐risk marginal croplands that are potentially suitable for growing cellulosic feedstock crops (e.g., switchgrass) in the US Great Plains (GP). Satellite‐derived growing season Normalized Difference Vegetation Index, a switchgrass biomass productivity map obtained from a previous study, US Geological Survey (USGS) irrigation and crop masks, and US Department of Agriculture (USDA) crop indemnity maps for the GP were used in this study. Our hypothesis was that croplands with relatively low crop yield but high productivity potential for switchgrass may be suitable for converting to switchgrass. Areas with relatively low crop indemnity (crop indemnity <$2 157 068) were excluded from the suitable areas based on low probability of crop failures. Results show that approximately 650 000 ha of marginal croplands in the GP are potentially suitable for switchgrass development. The total estimated switchgrass biomass productivity gain from these suitable areas is about 5.9 million metric tons. Switchgrass can be cultivated in either lowland or upland regions in the GP depending on the local soil and environmental conditions. This study improves our understanding of ecosystem services and the sustainability of cropland systems in the GP. Results from this study provide useful information to land managers for making informed decisions regarding switchgrass development in the GP.     

Modeling spatial dependence and economic hotspots in landowners’ willingness to supply bioenergy crops in the northeastern United States

This paper investigates the spatial heterogeneity of landowners’ willingness to supply three bioenergy crops: switchgrass, Miscanthus, and willow, in the northeastern United States. Spatial heterogeneity might arise for several reasons. For example, landowners closer to bioenergy processing plants might be more likely to be willing to supply bioenergy crops, and landowners who are more willing to supply bioenergy crops may be spatially clustered because they share similar land attributes, demographics, experiences, and/or values. Using high‐resolution GIS data related to the location of pellet plants utilizing bioenergy crops and survey data related to landowners’ characteristics including spatial location, we estimate a spatial probit model to explain the variation in individual‐specific reservation prices (RPs)—the feedstock price at which landowners become willing to supply a bioenergy crop. We find that respondents’ RP is lower the closer they live to their nearest pellet plant and spatial dependency is only present for switchgrass supply. We also identify three economic hotspots (areas with high potential supply and low RPs) for each bioenergy crop. We believe that bioenergy supply chains could be developed around these hotspots.     

pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage

ABSTRACT: BACKGROUND: The production of transgenic plants, either for the overproduction of the protein of interest, for promoter::reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming. RESULTS: We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1::DsRed marker and a CaMV::GUS cassette within the T-DNA and is useful for the production of promoter::GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1::DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia. CONCLUSIONS: The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

Development of a Gateway-compatible two-component expression vector system for plants

Genetic transformation of plants offers the possibility of functional characterization of individual genes and the improvement of plant traits. Development of novel transformation vectors is essential to improve plant genetic transformation technologies for various applications. Here, we present the development of a Gateway-compatible two-component expression vector system for Agrobacterium-mediated plant transformation. The expression system contains two independent plasmid vector sets, the activator vector and the reporter vector, based on the concept of the GAL4/UAS trans-activation system. The activator vector expresses a modified GAL4 protein (GAL4-VP16) under the control of specific promoter. The GAL4-VP16 protein targets the UAS in the reporter vector and subsequently activates reporter gene expression. Both the activator and reporter vectors contain the Gateway recombination cassette, which can be rapidly and efficiently replaced by any specific promoter and reporter gene of interest, to facilitate gene cloning procedures. The efficiency of the activator–reporter expression system has been assessed using agroinfiltration mediated transient expression assay in Nicotiana benthamiana and stable transgenic expression in Arabidopsis thaliana. The reporter genes were highly expressed with precise tissue-specific and subcellular localization. This Gateway-compatible two-component expression vector system will be a useful tool for advancing plant gene engineering.

     

Identifying potential areas for biofuel production and evaluating the environmental effects: a case study of the James River Basin in the Midwestern United States

Biofuels are now an important resource in the United States because of the Energy Independence and Security Act of 2007. Both increased corn growth for ethanol production and perennial dedicated energy crop growth for cellulosic feedstocks are potential sources to meet the rising demand for biofuels. However, these measures may cause adverse environmental consequences that are not yet fully understood. This study 1) evaluates the long‐term impacts of increased frequency of corn in the crop rotation system on water quantity and quality as well as soil fertility in the James River Basin and 2) identifies potential grasslands for cultivating bioenergy crops (e.g. switchgrass), estimating the water quality impacts. We selected the soil and water assessment tool, a physically based multidisciplinary model, as the modeling approach to simulate a series of biofuel production scenarios involving crop rotation and land cover changes. The model simulations with different crop rotation scenarios indicate that decreases in water yield and soil nitrate nitrogen (NO₃‐N) concentration along with an increase in NO₃‐N load to stream water could justify serious concerns regarding increased corn rotations in this basin. Simulations with land cover change scenarios helped us spatially classify the grasslands in terms of biomass productivity and nitrogen loads, and we further derived the relationship of biomass production targets and the resulting nitrogen loads against switchgrass planting acreages. The suggested economically efficient (planting acreage) and environmentally friendly (water quality) planting locations and acreages can be a valuable guide for cultivating switchgrass in this basin. This information, along with the projected environmental costs (i.e. reduced water yield and increased nitrogen load), can contribute to decision support tools for land managers to seek the sustainability of biofuel development in this region.     

Land‐use legacies regulate decomposition dynamics following bioenergy crop conversion

Land‐use conversion into bioenergy crop production can alter litter decomposition processes tightly coupled to soil carbon and nutrient dynamics. Yet, litter decomposition has been poorly described in bioenergy production systems, especially following land‐use conversion. Predicting decomposition dynamics in postconversion bioenergy production systems is challenging because of the combined influence of land‐use legacies with current management and litter quality. To evaluate how land‐use legacies interact with current bioenergy crop management to influence litter decomposition in different litter types, we conducted a landscape‐scale litterbag decomposition experiment. We proposed land‐use legacies regulate decomposition, but their effects are weakened under higher quality litter and when current land use intensifies ecosystem disturbance relative to prior land use. We compared sites left in historical land uses of either agriculture (AG) or Conservation Reserve Program grassland (CRP) to those that were converted to corn or switchgrass bioenergy crop production. Enzyme activities, mass loss, microbial biomass, and changes in litter chemistry were monitored in corn stover and switchgrass litter over 485 days, accompanied by similar soil measurements. Across all measured variables, legacy had the strongest effect (P < 0.05) relative to litter type and current management, where CRP sites maintained higher soil and litter enzyme activities and microbial biomass relative to AG sites. Decomposition responses to conversion depended on legacy but also current management and litter type. Within the CRP sites, conversion into corn increased litter enzymes, microbial biomass, and litter protein and lipid abundances, especially on decomposing corn litter, relative to nonconverted CRP. However, conversion into switchgrass from CRP, a moderate disturbance, often had no effect on switchgrass litter decomposition parameters. Thus, legacies shape the direction and magnitude of decomposition responses to bioenergy crop conversion and therefore should be considered a key influence on litter and soil C cycling under bioenergy crop management.