木质素的遗传操纵减少了内在反抗以及提高柳枝稷的乙醇生产能力.docx

木质素的遗传改造以减少了顽抗以及提高柳枝稷的乙醇生产能力

摘录
Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements.柳枝稷在美国是一种先进的生物能源原料，因为它是一种本地的、高产的、常年有的、具有广泛的种植范围以及低农艺输入要求Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass.生物质转化的研究开发以及工艺生产乙醇和其他生物燃料，但它们仍然昂贵，这主要是因为生物质的固有顽抗。We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical (≤180 °C), enzymatic, and microbial recalcitrance.我们在这里展示的柳枝稷能产生遗传修饰的表型正常的植物，减少了热化学（≤180°C），酶和微生物的顽抗。Down-regulation of the switchgrass caffeic acid O -methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes.柳枝咖啡酸O-甲基转移酶基因的下调温和降低木质素含量，降低紫丁香：愈创木基木质素单体比，提高饲料质量。并且，最重要的是，增加乙醇产量提高到38％，使用传统的生物质发酵过程。The down-regulated lines require less severe pretreatment and 300–400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast.降低要求不太重要的预处理和用量相当于产品产量低300-400％的纤维素酶同步糖化发酵用酵母。Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass.此外，使用不添加酶的嗜热梭菌发酵稀酸预处理转基因比未修改柳枝稷表现出更好的产品产量。Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly.因此，转基因柳枝稷固有顽抗明显的减少，可能生物质发酵衍生的燃料和化学品的加工费用也明显降低。另外，这种修饰的转基因柳枝稷线应在相同的工艺条件下，每公顷产量显着更多的化学品。
木质纤维素生物质是丰富的，可再生的原料，可以通过发酵转化为可运输的液体燃料和其他化学品。Cellulosic ethanol is a promising near-term technological option to reduce transportation sector greenhouse gas emissions ( 1 ).纤维素乙醇是一种很有前途的短期技术选项，以减少运输部门的温室气体排放量。Because lignocellulosic biomass is made up of the complex structures of cellulose, hemicellulose, and lignin, such feedstock is highly recalcitrant to bioconversion of its carbohydrates into ethanol compared with starch ( 2 , 3 ).由于木质纤维素生物质是由纤维素，半纤维素和木质素等构成，例如原料是高度顽抗淀粉相比，其转化为乙醇的碳水化合物的生物转化。Current biomass fermentation processes for fuels and chemicals have a relatively high cost primarily because of this recalcitrance, which in turn has limited commercialization of biomass ethanol ( 4 ).当前生物质发酵过程作为燃料和化学品有相对较高的成本主要是由于这种顽抗，进而限制了商业化的生物质乙醇。To achieve sustainable energy production, it is necessary to overcome the chemical and structural properties of biomass that inhibit its deconstruction in dedicated bioenergy crops ( 5 ).为了实现可持续的能源生产，必须克服化学和结构性能的生物质，抑制其解构在专用的生物能源作物上。
木质生物质转化乙醇是一个三步的过程，涉及预处理，其次是多糖水解单糖其次是糖发酵乙醇。存在木质素在细胞壁负面影响这些转换步骤。实验自然变异在紫花苜蓿、柳枝稷和高粱小子虉草，表明降低木质素含量提高体外酶水解。木质素改性途径在苜蓿转基因线生成增加酶糖释放与木质素下调基本上成正比的程度。尽管转基因的方法已经被用来描述木质素生物合成途径和提高细胞壁的特征，大多数的研究已经进行了与双子叶植物物种在上下文的合成饲料和纸制浆，只有有限的信息可用于多年生单子叶植物。迄今为止，还没有报告关于发酵的改良植物乙醇生产在任何转基因多年生生物燃料作物。
Switchgrass ( Panicum virgatum L.) is a dedicated energy crop identified by the US Department of Energy ( 15 ).柳枝稷（ Panicum virgatum L。）是由美国能源部确定一个专门的能源作物。It is native to the United States and is a productive perennial C4 species, with a broad cultivation range, that requires relatively minimal agronomic inputs as a biofuel crop ( 15 , 16 ).它是原产于美国，是一个常年生产的C4植物，具有广泛的种植范围，需要相对较少的作为一种生物燃料作物的农艺输入。Field studies demonstrated that switchgrass grown and managed as a biomass crop produces 540% more renewable energy than energy consumed in its production and has significant environmental benefits ( 16 ).实地研究表明，柳枝稷的种植和管理作为生物质作物在它的生产时比它消耗的能量多产生540％的可再生能源的能量，并具有显着的环境效益。Genetic improvement of switchgrass to reduce intrinsic recalcitrance to fermentative bioprocessing would improve biofuel and chemical production processes and have a profound positive impact on the nascent bioenergy industry.柳枝稷的遗传改良，以减少内在顽抗发酵生物加工将提高生物燃料和化工生产过程和对新生的生物能源行业产生深远的积极的影响。
We show here that down-regulation of the caffeic acid 3- O -methyltransferase ( COMT ) gene in the lignin pathway leads to the generation of transgenic switchgrass plants with a normal growth phenotype that have reduced lignin content, altered lignin composition, improved forage quality, increased saccharification efficiency, and increased ethanol production yield on substrate compared with the controls.我们在这里展示咖啡酸3 - O-甲基转移酶 （COMT）基因中的木质素途径下调导致代转基因柳枝植物正常生长型，降低木质素含量，改变木质素组成，提高饲草质量与对照组相比，糖化效率增加，在基板上的乙醇生产产量增加。Moreover, the transgenic plant materials require less severe pretreatment and much lower cellulase dosages to obtain ethanol yields equivalent to yields in controls.此外，转基因植物材料要求不太高的预处理和纤维素酶剂量低得多获得乙醇产量相当于对照的产量。These transgenic switchgrass lines and the approach are valuable for developing improved cultivars of biofuel crops.这些转基因柳枝稷的线条和方法是有价值的对于发展生物燃料作物的品种改良。

结果
柳枝COMT基因调控
在Based on earlier results of lignin modification in alfalfa and other species ( 14 , 17 , 18 ), we chose to down-regulate the COMT gene in switchgrass using the widely distributed variety “Alamo.” We constructed a cDNA library from 2-mo-old switchgrass seedlings and isolated a COMT cDNA containing a 1,086-bp ORF.紫花苜蓿和其他物种的早期成果的基础上的木质素改性，我们选择调控COMT基因柳枝使用广泛分布的各种“Alamo”2个月，我们构建了cDNA文库老柳枝苗和孤立一个COMT的基因包含一个1，086 bp的ORF。Partial sequences of the ORF were used to construct an RNAi vector, which was transferred into Agrobacterium tumefaciens strain EHA105, and transgenic switchgrass plants were obtained by Agrobacterium -mediated transformation of embryogenic calli.部分序列的ORF被用来构建RNAi载体转入农杆菌菌株EHA105，通过农杆菌介导的胚性愈伤组织，获得转基因柳枝植物。RT-PCR and quantitative real-time PCR analyses of selected transgenic plants revealed significantly reduced transcript abundance ( Fig. 1 A and B ) in most of the lines, with more than 90% reduction observed in the lines T0-2 and T0-3 ( Fig. 1 B ). RT-PCR和实时定量PCR分析所选的转基因植物显示在(图1 A and B)大多数的线条上，减少90％以上观察到的线条T0-2和T0-3转录显着降低（ 图1中 ）（ 图1B ）。To determine whether reduced COMT transcript resulted in the reduction of COMT enzyme activity, crude enzyme extracts prepared from the transgenic and control plants were assayed with two preferred COMT substrates, 5-OH coniferaldehyde and caffeyl aldehyde ( Fig. 1 C ). ，以确定是否转移酶转录降低导致的COMT酶的活性，减少粗酶提取物制备的转基因和对照植株检测与两个优选的转移酶的底物，5-OH coniferaldehyde和caffeyl醛（ 图1 C ）。Except for line T0-9, all transgenic plants showed significant reduction in COMT enzyme activity.除线T0-9，所有的转基因植物显示COMT酶的活性显着降低。The most strongly down-regulated lines, T0-2 and T0-3, had 22–25% and 25–28% residual enzyme activity, respectively, depending on the substrates used ( Fig. 1 C ).最强烈的下调线，T0-2和T0-3，有22-25％和25-28％的残留酶活性，分别根据所用基片（ 图1 C ）。T0 lines T0-2, T0-3, and a moderately down-regulated line T0-12 were chosen for further analysis. T0线T0-2，T0-3，和适度下调线T0-12被选择用于进一步分析。Because of the outcrossing requirement of switchgrass, these lines were outcrossed with a wild-type plant to obtain progeny seeds designated as T1 lines.由于异交要求柳枝稷，这些线路与野生型植物远缘杂交，取得指定为T1线路的后代种子。Both COMT RNAi-positive and -negative (null segregant) plants were identified from the progeny of each cross, and the negative plants were used as controls for analyses of the corresponding T1 transgenic plants.这两种COMT RNAi的阳性和阴性（分离子空）确定从每个交叉的后代植物，使用负的植物作为对照进行分析，对应于T1的转基因植物。
COMT抑制木质素含量、成分和植物生长的影响
We examined lignin content and lignin monomer composition in whole tillers (including stem, leaf, and sheath) and stems of transgenic switchgrass.我们研究木质素含量和木质素单体组成在整个分蘖（包括茎，叶，鞘）和来源于转基因柳枝稷。In the T0 generation, transgenic lines T0-2, T0-3, and T0-12 showed significant reduction in their acetyl bromide (AcBr) lignin content (12.2% for T0-2, 14.7% for T0-3, and 6.4% for T0-12), syringyl (S) and guaiacyl (G) lignin monomer content, and S/G ratios for whole tillers ( Table 1 ).在T0代转基因株系T0-2，T0-3，和T0-12表明乙酰溴（ACBR）木素含量显着降低（12。2％T0-2，T0-3 14。7％，及6。4％为T0-12），紫丁香（S）和愈创木基（G）的的木质素单体含量，S / G的比值为全分蘖（ 表1 ）。Even after outcrossing with wild-type plants, the T1 generation of the two most down-regulated lines T1-2 and T1-3, showed reductions in both AcBr lignin, at 11.4–13.4%, and S and G lignin content similar to the reductions seen in the respective T0 lines.与野生型植物异花授粉后，即使T1代的两个最下调线，T1-2和T1-3，显示减少两个ACBR木质素，在11。4-13。4％，和S和G木质素含量类似于在各自的T0线看到的减少。The resulting S/G ratio was essentially identical in the T1 and T0 lines, at 0.37–0.40, versus 0.69–0.71 in controls ( Table 1 ).将所得的S / G的比值是在T1和T0线基本上是相同的，为0。37-0。40，而对照组（ 表1 ） 0。69-0。71 。The stem material had similar levels of reduction in lignin content and a higher S/G ratio ( Table S1 ).干材料中的木质素含量和较高的S / G的比值（ 表S1 ）有相似水平的减少。
复杂细胞壁多糖的组合物，进行评价，以确定潜在的更广泛影响COMT下调。The T0 and T1 lines exhibited small variations in the levels of cellulose, at −3% to −5% for T0 lines and +3% for T1 lines compared with controls ( Table S2 ). T0和T1线展出纤维素水平的微小变化，在-3％至-5％，与对照组相比（ 表S2 ）为T0线和+3％T1线路。The cellulose crystallinity index (CrI) and degree of polymerization (DP) of the T1-2 and T1-3 lines were essentially identical to those of controls ( Fig. S1 A and B ).纤维素的结晶度指数（CRI）和聚合度（DP）的T1-2和T1-3线基本上是相同的对照组（ S1图A和B ）。Similarly, the stem material for T0 and T1 transgenic lines showed a −1% to +3% change in cellulose content compared with controls ( Table S3 ).同样，T0和T1转基因株系的茎材料显示-1％+3％的纤维素含量的变化与对照组相比（ 表S3 ）。Apparently, the reduced lignin content has minor or negligible impact on cellulose content or structure.显然，降低木质素含量有轻微或微不足道的影响纤维素内容或结构。