杨树茎木质素的RNAi调控及糖化特性分析
摘要
随着人们对环境质量和生活品质的重视以及保障能源安全的需求,生物质能源越来越受到公众和科学界的关注。考虑到生物柴油生产的高成本,生物乙醇成为全球最广泛利用的生物燃料。但由于目前生产生物乙醇的原料主要来自于谷物等粮食作物,由此牵涉到到全球粮价上涨和粮食恐慌,加之其有限的能源产量,使得通过粮食发酵生产燃料乙醇的规模化发展受到限制。在保证粮食安全的前提下解决能源危机问题,必然聚焦于林木生物质能源。木质纤维素是地球上数量最大的一种可再生资源,发展经济有效而环境友好的木质纤维素燃料乙醇技术,已成为世界生物能源科技发展的趋势。针对我国国情,发展以木质纤维生物转化乙醇为重点的生物能源技术,建立以能源林业为依托的原料保障模式,可以实现“不与人争粮”、“不与粮争地”的目的,兼得经济、生态、环保、社会多重效益。作为木本模式植物的杨树是全球广泛栽培的重要造林树种之一,我国杨树资源丰富,是世界上人工林面积最大的国家。杨树分布广、实用性强、无性繁殖能力强,加之其速生丰产、遗传背景清楚等优势,无可争议地成为林木生物质能源研究的首选。林木生物质能源发展面临的最大技术难题是木质纤维素的水解。由于木质素与纤维素的紧密结合和对其保护作用是导致纤维素资源利用的主要障碍。降低木质素含量或改变其结构,将有利于纤维素的解聚和糖转化效率的提高。
本研究根据前人研究成果,选定毛白杨香豆酰莽草酸/奎宁酸羟化酶C3H、肉桂酸-4-羟基化酶C4H和羟基肉桂酰辅酶A:莽草酸/奎尼酸羟基肉桂酰转移酶HCT基因为调控目标,通过RT-PCR的方法得到毛白杨C3H1、C4H1、HCT1和HCQ3基因的cDNA全长序列,C3H1基因的cDNA完整的开放读码框(ORF)共编码508个氨基酸,HCT1编码432个氨基酸,HCQ3编码431个氨基酸,C4H1基因编码505个氨基酸。依此为基础构建了毛白杨C3H1、C4H1、HCT1和HCQ3基因的RNAi抑制表达载体pBIRNAi-C3H1R-i-C3H1L、pBIRNAi-C4H1R-i-C4H1L、pBIRNAi-HCT1R-i-HCT1L和pBIRNAi-HCQ3R-i-HCQ3L。进一步利用Gateway技术和已构建的RNAi抑制表达载体,构建了含有毛白杨C3H1、C4H1、HCT1和HCQ3基因多基因不同组合发夹结构的共15个RNAi抑制表达载体,以用于农杆菌介导的杨树遗传转化。
通过根癌农杆菌介导的叶盘法转化银腺杨无性系84K,经农杆菌培养、侵染、共培养、选择培养、继代选择培养和生根培养后获得转基因阳性植株。经NPT-II基因和目标基因片段的PCR鉴定后移栽温室,经低温诱导后温室扦插繁殖,最终获得转C3H1基因RNAi抑制表达载体pBIRNAi-C3H1R-i-C3H1L共8个株系25个单株、转HCT1基因RNAi抑制表达载体pBIRNAi-HCT1R-i-HCT1L的15个株系33个单株和转HCQ3基因RNAi抑制表达载体pBIRNAi-HCQ3R-i-HCQ3L的7个株系25个单株。
扦插繁殖后的转基因阳性植株和对照植株新萌枝条通过Realtime PCR检测目标基因转录表达量,转基因株系323、325和322中C3H1基因表达量较野生型植株分别下调了89.04%、82.22%和68.38%,312、308、502和307各株系HCQ3基因转录量比野生型植株的平均表达量下调了67.64%、56.35%、49.88%和45.05%。
茎横切片组化染色和显微结构观察结果表明,基因表达受抑制的转基因植株次生木质部细胞层数增多,细胞较小,导管壁塌陷和部分细胞次生壁的不规则区域性加厚,反映出转基因植株木质部发育和木质素沉积方式发生了改变。
木质素、纤维素含量测定结果表明,转基因植株木质素含量的降低与目标基因的转录表达量下调总体相符,木质素降低的转基因植株表现出较高的纤维素含量,C3H1转基因植株木质素含量平均降低23.00%,最高降低了39.71%;HCQ3转基因植株木质素含量平均降低了37.65%,最高达58.19%。苯酚-硫酸法总糖含量、糖转化效率测定与HPLC法可溶性总糖、单糖含量检测结果表明,木质素含量的降低可导致细胞可溶性糖含量的增加及纤维素的糖转化效率的提高。C3H1和HCQ3转基因植株酸前处理效率较对照平均提高了62.41%和119.18%,酸前处理后酶解效率最高提高了51.74%;说明由于木质素含量的降低可能引发了细胞中可溶性糖和纤维素的代偿性增加,同时由于木质素的减少,减轻了对纤维素的束缚作用,游离的纤维素增多,纤维素复合酶对纤维素的可及度增大,使得酶解糖化效率显著提高。未经酸前处理的转基因植株糖化效率明显高于对照植株,甚至达到或高于经酸前处理后再酶处理的糖化效率,说明木质素含量可能是阻碍细胞壁糖化作用的主要因素,通过RNAi技术抑制木质素合成关键酶基因表达,获得低木质素含量、高糖转化效率的转基因植株,为生物质能源杨树的分子育种和高效利用林木生物质能源提供了理论依据和技术支持。
Biofuels are gaining increased public and scientific attention, driven by the desires to improve air quality and secure energy supply. Considering high-cost in biodiesel production, ethanol becomes the most promising biofuel. Ethanol from food stocks has implications of world food prices and limited energy yield, thus the technology has led to the development of cellulosic ethanol. To relieve the energy crisis under food security, forest bioenergy is thus inevitably focused on. Lignocellulose is the largest renewable resource on earth. In China, if the pipeline of lignocellulose bioconversion to ethanol and the energy feedstock based on foresty are established, the purpose of "not competing with food" and "not competing with lands" can be achieved with economic, environmental and social benefits. Poplar, as a model tree species, is widely cultivated globally as the important plantation trees, and China is the world's largest in poplar plantation. The poplar genus is widely distributed, together with its asexual reproduction ability, fast growth and high yield, could minimize the competition between biofuels and food crops. In addition, the sequenced genome of ploplar exhibits more attractive for advanced breeding as an alternative biofuel source. However, efficient hydrolysis of lignocellulose is the biggest technical challenge on forest bionergy development because lignin is a major limitation for converting lignocellulose to ethanol in poplar.
We are attempting to genetically modify poplar to decrease lignin content in order to enhance fermenTab. sugars which can be converted to ethanol. To do this, we targeted at coumaroyl shikimate 3-hydroxylase ( C3H ) , cinnamate 4-hydroxylase ( C4H ) and hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase(HCT)involved in lignin biosynthesis using RNAi technique. Four full gene-length cDNAs of C3H1, C4H1, HCT1 and HCQ3 in Populus tomentosa Carr. were cloned using in silico and molecular technique. The open reading frame of C3H1, HCT1, HCQ3 and C4H1 gene encodes a peptide of 508, 432, 431 and 505 amino acids respectively. RNAi vectors expressing ds-RNA for each of them were constructed. Additional fifteen multi-gene fusion RNAi constucts with different combinations of the four genes used for transformation were obtained by employing a Gateway-based platform(pBIRNAi-C3H1R-i-C3H1L、pBIRNAi-C4H1R-i-C4H1L、pBIRNAi-HCT1R-i-HCT1L and pBIRNAi-HCQ3R-i-HCQ3L) Poplar was transformed via the Agrobactria-mediated leaf-disc method. 30 independent transgenic poplar lines harboring the three gene RNAi constructs were obtained and vegetatively propagated by cutting for each lines in the greenhouse.
The kanamycin-resistant seedlings were subjected to further analyses of gene expression by real-time PCR. The transcription level of C3H1 and HCQ3 in both non- and transgenic lines were analyzed by real-time PCR. 89.04%, 82.22% and 68.38% reduction were obtained in the C3H1 RNAi inhibition lines 323, 325 and 322 respectively in comparison with the non-transgenic controls, and 67.64%, 56.35%, 49.88% and 45.05% reduction were obtained in the HCQ3 RNAi inhibition lines 312, 308, 502 and 307.
Stem cross-section staining and microstructure observations showed that cell layers of the secondary xylem in transgenic plants were increased, but the cell became smaller with cell wall collapsed and irregular thickened secondary wall. It indicated that the xylem development and lignin deposition pattern in transgenic plants were changed. Lignin and cellulose content test showed that transgenic plants with reduced lignin content generally in accordance with the transcript level of the target gene. Lignin reduction in transgenic plants also showed higher cellulose content.
To determine relationships between lignin content and the efficiency of chemical/enzymatic saccharification, stem material with modified lignin content were tested. Solubilized total sugars in extractive free cell wall residues and hydrolyzates were estimated spectrophotometrically using the phenol-sulfuric acid assay. Monomeric sugars in hydrolysates from acid pre-treatment and the glucose and xylose contents of enzymatic hydrolysates were determined by HPLC. Plants with the least lignin had the highest total carbohydrate levels in untreated biomass, re?ecting compensation for the reduction in lignin level on a mass balance basis. After 72 h incubation, saccharification efficiency was higher in C3H and HCT reduced lines compared with controls. More than 90% of the released sugar from most lines was glucose, indicating enzymatic hydrolysis of cellulose. Enzymatic hydrolysis released more xylose from transgenic lines than from control lines, suggesting that lignin modification increases the accessibility of residual hemicellulose to degradative enzymes.
This study tested the effectiveness of reduction of lignin content by RNAi strategy using several key genes and their fusion constructs. The results indicated that lignin is probably the major factor in recalcitrance of cell walls to saccharification. Moreover, it demonstrated that genetic reduction of lignin content effectively overcame cell wall recalcitrance to bioconversion. This approach could obviate the need for acid pretreatment, as indicated by that the saccharification efficiency of untreated biomass of the 312 and 323 line were even greater than that of control plants with pretreatment. The genes targeted in the present work thus would be the candidates for improving saccharification in other bioenergy plants.
本研究根据前人研究成果,选定毛白杨香豆酰莽草酸/奎宁酸羟化酶C3H、肉桂酸-4-羟基化酶C4H和羟基肉桂酰辅酶A:莽草酸/奎尼酸羟基肉桂酰转移酶HCT基因为调控目标,通过RT-PCR的方法得到毛白杨C3H1、C4H1、HCT1和HCQ3基因的cDNA全长序列,C3H1基因的cDNA完整的开放读码框(ORF)共编码508个氨基酸,HCT1编码432个氨基酸,HCQ3编码431个氨基酸,C4H1基因编码505个氨基酸。依此为基础构建了毛白杨C3H1、C4H1、HCT1和HCQ3基因的RNAi抑制表达载体pBIRNAi-C3H1R-i-C3H1L、pBIRNAi-C4H1R-i-C4H1L、pBIRNAi-HCT1R-i-HCT1L和pBIRNAi-HCQ3R-i-HCQ3L。进一步利用Gateway技术和已构建的RNAi抑制表达载体,构建了含有毛白杨C3H1、C4H1、HCT1和HCQ3基因多基因不同组合发夹结构的共15个RNAi抑制表达载体,以用于农杆菌介导的杨树遗传转化。
通过根癌农杆菌介导的叶盘法转化银腺杨无性系84K,经农杆菌培养、侵染、共培养、选择培养、继代选择培养和生根培养后获得转基因阳性植株。经NPT-II基因和目标基因片段的PCR鉴定后移栽温室,经低温诱导后温室扦插繁殖,最终获得转C3H1基因RNAi抑制表达载体pBIRNAi-C3H1R-i-C3H1L共8个株系25个单株、转HCT1基因RNAi抑制表达载体pBIRNAi-HCT1R-i-HCT1L的15个株系33个单株和转HCQ3基因RNAi抑制表达载体pBIRNAi-HCQ3R-i-HCQ3L的7个株系25个单株。
扦插繁殖后的转基因阳性植株和对照植株新萌枝条通过Realtime PCR检测目标基因转录表达量,转基因株系323、325和322中C3H1基因表达量较野生型植株分别下调了89.04%、82.22%和68.38%,312、308、502和307各株系HCQ3基因转录量比野生型植株的平均表达量下调了67.64%、56.35%、49.88%和45.05%。
茎横切片组化染色和显微结构观察结果表明,基因表达受抑制的转基因植株次生木质部细胞层数增多,细胞较小,导管壁塌陷和部分细胞次生壁的不规则区域性加厚,反映出转基因植株木质部发育和木质素沉积方式发生了改变。
木质素、纤维素含量测定结果表明,转基因植株木质素含量的降低与目标基因的转录表达量下调总体相符,木质素降低的转基因植株表现出较高的纤维素含量,C3H1转基因植株木质素含量平均降低23.00%,最高降低了39.71%;HCQ3转基因植株木质素含量平均降低了37.65%,最高达58.19%。苯酚-硫酸法总糖含量、糖转化效率测定与HPLC法可溶性总糖、单糖含量检测结果表明,木质素含量的降低可导致细胞可溶性糖含量的增加及纤维素的糖转化效率的提高。C3H1和HCQ3转基因植株酸前处理效率较对照平均提高了62.41%和119.18%,酸前处理后酶解效率最高提高了51.74%;说明由于木质素含量的降低可能引发了细胞中可溶性糖和纤维素的代偿性增加,同时由于木质素的减少,减轻了对纤维素的束缚作用,游离的纤维素增多,纤维素复合酶对纤维素的可及度增大,使得酶解糖化效率显著提高。未经酸前处理的转基因植株糖化效率明显高于对照植株,甚至达到或高于经酸前处理后再酶处理的糖化效率,说明木质素含量可能是阻碍细胞壁糖化作用的主要因素,通过RNAi技术抑制木质素合成关键酶基因表达,获得低木质素含量、高糖转化效率的转基因植株,为生物质能源杨树的分子育种和高效利用林木生物质能源提供了理论依据和技术支持。
Biofuels are gaining increased public and scientific attention, driven by the desires to improve air quality and secure energy supply. Considering high-cost in biodiesel production, ethanol becomes the most promising biofuel. Ethanol from food stocks has implications of world food prices and limited energy yield, thus the technology has led to the development of cellulosic ethanol. To relieve the energy crisis under food security, forest bioenergy is thus inevitably focused on. Lignocellulose is the largest renewable resource on earth. In China, if the pipeline of lignocellulose bioconversion to ethanol and the energy feedstock based on foresty are established, the purpose of "not competing with food" and "not competing with lands" can be achieved with economic, environmental and social benefits. Poplar, as a model tree species, is widely cultivated globally as the important plantation trees, and China is the world's largest in poplar plantation. The poplar genus is widely distributed, together with its asexual reproduction ability, fast growth and high yield, could minimize the competition between biofuels and food crops. In addition, the sequenced genome of ploplar exhibits more attractive for advanced breeding as an alternative biofuel source. However, efficient hydrolysis of lignocellulose is the biggest technical challenge on forest bionergy development because lignin is a major limitation for converting lignocellulose to ethanol in poplar.
We are attempting to genetically modify poplar to decrease lignin content in order to enhance fermenTab. sugars which can be converted to ethanol. To do this, we targeted at coumaroyl shikimate 3-hydroxylase ( C3H ) , cinnamate 4-hydroxylase ( C4H ) and hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase(HCT)involved in lignin biosynthesis using RNAi technique. Four full gene-length cDNAs of C3H1, C4H1, HCT1 and HCQ3 in Populus tomentosa Carr. were cloned using in silico and molecular technique. The open reading frame of C3H1, HCT1, HCQ3 and C4H1 gene encodes a peptide of 508, 432, 431 and 505 amino acids respectively. RNAi vectors expressing ds-RNA for each of them were constructed. Additional fifteen multi-gene fusion RNAi constucts with different combinations of the four genes used for transformation were obtained by employing a Gateway-based platform(pBIRNAi-C3H1R-i-C3H1L、pBIRNAi-C4H1R-i-C4H1L、pBIRNAi-HCT1R-i-HCT1L and pBIRNAi-HCQ3R-i-HCQ3L) Poplar was transformed via the Agrobactria-mediated leaf-disc method. 30 independent transgenic poplar lines harboring the three gene RNAi constructs were obtained and vegetatively propagated by cutting for each lines in the greenhouse.
The kanamycin-resistant seedlings were subjected to further analyses of gene expression by real-time PCR. The transcription level of C3H1 and HCQ3 in both non- and transgenic lines were analyzed by real-time PCR. 89.04%, 82.22% and 68.38% reduction were obtained in the C3H1 RNAi inhibition lines 323, 325 and 322 respectively in comparison with the non-transgenic controls, and 67.64%, 56.35%, 49.88% and 45.05% reduction were obtained in the HCQ3 RNAi inhibition lines 312, 308, 502 and 307.
Stem cross-section staining and microstructure observations showed that cell layers of the secondary xylem in transgenic plants were increased, but the cell became smaller with cell wall collapsed and irregular thickened secondary wall. It indicated that the xylem development and lignin deposition pattern in transgenic plants were changed. Lignin and cellulose content test showed that transgenic plants with reduced lignin content generally in accordance with the transcript level of the target gene. Lignin reduction in transgenic plants also showed higher cellulose content.
To determine relationships between lignin content and the efficiency of chemical/enzymatic saccharification, stem material with modified lignin content were tested. Solubilized total sugars in extractive free cell wall residues and hydrolyzates were estimated spectrophotometrically using the phenol-sulfuric acid assay. Monomeric sugars in hydrolysates from acid pre-treatment and the glucose and xylose contents of enzymatic hydrolysates were determined by HPLC. Plants with the least lignin had the highest total carbohydrate levels in untreated biomass, re?ecting compensation for the reduction in lignin level on a mass balance basis. After 72 h incubation, saccharification efficiency was higher in C3H and HCT reduced lines compared with controls. More than 90% of the released sugar from most lines was glucose, indicating enzymatic hydrolysis of cellulose. Enzymatic hydrolysis released more xylose from transgenic lines than from control lines, suggesting that lignin modification increases the accessibility of residual hemicellulose to degradative enzymes.
This study tested the effectiveness of reduction of lignin content by RNAi strategy using several key genes and their fusion constructs. The results indicated that lignin is probably the major factor in recalcitrance of cell walls to saccharification. Moreover, it demonstrated that genetic reduction of lignin content effectively overcame cell wall recalcitrance to bioconversion. This approach could obviate the need for acid pretreatment, as indicated by that the saccharification efficiency of untreated biomass of the 312 and 323 line were even greater than that of control plants with pretreatment. The genes targeted in the present work thus would be the candidates for improving saccharification in other bioenergy plants.
引文
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