植物肉桂醇脱氢酶及其基因研究进展

肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase,CAD)作为植物次生代谢特别是木质素合成的关键酶,与植物生长发育和抵御病原菌入侵关系密切,研究CAD基因表达调控及其与组织木质化的关系具有重要的植物生理学意义.该文综述了植物CAD的蛋白特征、酶学性质、基因分布和分类、基因结构和表达调控以及CAD表达与木质素合成的关系,为研究CAD在植物生长发育和抗病中的作用提供理论指导.     

高等植物赤霉素2-氧化酶基因的克隆、表达及其调控

赤霉素(GA)是一类重要的植物激素,对高等植物整个生命周期的生长发育起关键作用。调控赤霉素生物合成和代谢途径中的关键酶基因的表达可以控制植物体内赤霉素的含量。GA2-氧化酶是调节赤霉素合成和代谢的关键酶之一,使活性GA失活。本文主要对GA2-氧化酶基因的克隆、表达调控及其在植物基因工程中的应用等方面进行综述,为通过基因工程技术调控植物体内活性赤霉素的含量从而得到改良品种提供思路。     

西瓜嫁接体发育中木质素合成及代谢相关酶活性的变化

研究了西瓜/葫芦嫁接体发育过程中砧木和接穗部分木质素含量及其代谢相关酶的活性变化.结果表明,嫁接体发育过程中木质素生物合成加快,砧木及接穗部分的过氧化物酶(POD)、过氧化氢酶(CAT)、苯丙氨酸解胺酶(PAL)、肉桂醇脱氢酶(CAD)等的活性均明显高于对照,接穗和砧木中POD活性在15 d内持续升高,H2O2含量和CAT活性于嫁接后9~12 d出现高峰,砧木中PAL于9 d时有活性高峰而接穗一直保持较高活性而没有活性高峰,木质素含量和CAD活性持续增长,葫芦砧木木质素的代谢水平高于西瓜接穗.     

不同苹果品种对褐斑病抗性的鉴定及抗性生理研究

对42个苹果栽培品种叶片褐斑病进行了田间发病状况调查与抗性鉴定,分析了叶片气孔密度和大小与抗病性之间的关系,同时研究了不同抗性品种离体叶片接种病原菌后超氧化物歧化酶（SOD）、多酚氧化酶（vPo）、过氧化物酶（POD）、苯丙氨酸解氨酶（PAL）活性和木质素含量的差异变化。依照抗性分级标准,供试材料中抗病品种有14个（其中高抗品种2个）,感病品种有28个（其中高感品种7个）;叶片气孔密度与病情指数之间存在显著正相关,相关系数r=0．683;叶片接种后,诱导了4种酶活性和木质素含量的升高,抗病和感病品种的SOD和PP0活性无显著差异,而抗病品种的POD和PAL活性以及木质素含量显著高于感病品种。苹果叶片的气孔密度、POD和PAL的活性以及木质素含量与褐斑病抗性有关。     

棉花漆酶基因在转基因新疆杨中的表达及其对木质素合成的影响

以新疆杨叶柄为外植体,利用农杆菌法将棉花漆酶基因GaLAC1导入新疆杨.PCR,Soutllern杂交证明外源基因已经整合到杨树基因组中.漆酶活性分析表明转基因植株中漆酶活性较非转基因对照显著提高.与对照植株相比,转基因新疆杨茎段中总木质素的含量有不同程度的增加,最高达21.5%.木质素的组织化学染色进一步证实了GaLAC1的过量表达能够导致转基因植株中总木质素含量的增加.实验结果表明GaLAC1参与了植物体内木质素的合成,这是首次成功利用转基因植物证实植物漆酶基因参与木质素合成的报道.     

矿质营养缺乏对黄瓜生理生化特性的影响

通过营养液栽培,研究不同矿质营养的缺乏对黄瓜叶片组织生理生化特性的影响。结果表明,缺铜对植株生理影响最大,在各处理中,叶片组织的可溶性糖含量最低,木质素含量也处于较低水平,而相对电导度则最高,所测定的4种保护酶(POD、PPO、PAL、SOD)活性也最低。其次是锌,缺锌造成了相对电导度的增加,可溶性糖含量和木质素含量的下降,除PAL活性外,其余3种酶活性也处于次低的位置。缺锰主要导致了叶片组织可溶性糖含量、木质素含量以及PAL活性的较大幅度的下降。缺钙主要降低了叶片组织中可溶性糖含量和SOD活性。比较而言,硼和铁的缺乏影响较小。     

谷胱甘肽对采后石刁柏木质化和食用品质的影响

在(24±1)℃条件下,谷胱甘肽(GSH)可显著抑制采后石刁柏木质素合成前体总酚的含量及与木质素合成相关的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性上升,延缓叶绿素、可溶性糖、可溶性蛋白和核酸的降解,降低活性氧和木质素含量,从而保持石刁柏的鲜嫩品质.     

转PcLIPH8基因水稻的构建及其相关表型分析

利用黄孢原毛平革菌木质素过氧化物酶基因(PcLIPH8),构建植物双元表达载体35S∷PcLIPH8,并遗传转化水稻野生型品种Kitaake,对转基因水稻进行分子鉴定、酶活及木质素含量测定、表型观察等分析。结果表明：(1) 成功构建了植物双元表达载体35S∷PcLIPH8,获得3个独立的转PcLIPH8水稻株系,但在苗期转基因水稻与野生型对照无明显表型差异。(2) 酶活及木质素含量测定结果表明,转基因水稻的木质素过氧化物酶活性增加3.06%~5.07%,而木质素含量显著低于野生型对照,苗期降低11.44%~14.97%,成熟期降低13.83%~20.05%。(3) 成熟期表型分析表明,转基因水稻较野生型对照的株高增加了28.37%~39.78%,穗谷粒数增多110%~120%,生物量增大18.61%~22.97%,而千粒重减小12.86%~13.34%,谷粒长度变短6.67%~7.15%。该研究结果为利用PcLIPH8基因降低木质素含量,提高生物产量,从而改善植物品质奠定了前期基础。     

橄榄果实发育过程中细胞壁物质和相关酶活性变化

为了解橄榄(Canarium album)果实质地差异形成的原因,以鲜食型橄榄‘清榄1号’和加工型橄榄‘长营’为材料,对果实发育过程中细胞壁物质含量和相关酶活性进行了测定。结果表明,随着橄榄果实的成熟,‘清榄1号’较‘长营’维持较高的果胶甲酯酶(PME)活性,促进了果胶的水解,离子型果胶(ISP)含量较高而共价型果胶(CSP)含量较低。2个橄榄品种纤维素含量均较高,‘清榄1号’果实的半纤维素含量低于‘长营’。‘清榄1号’木质素含量低于‘长营’,较高的苯丙氨酸解氨酶(PAL)和过氧化物酶(POD)活性促进了木质素含量的增加。因此,ISP、CSP、半纤维素和木质素含量的不同可能是2个橄榄品种果实质地差异形成的原因。     

苎麻CCoAOMT基因cDNA反义转化模式烟草'WS38'

苎麻咖啡酰辅酶A氧甲基转移酶(CCoAOMT)是其木质素合成过程的一种关键酶,运用克隆的该酶基因cDNA及植物表达载体pBI121、pWM101,分别构建了35S启动子控制的苎麻CCoAOMT基因反义cDNA基因质粒(pBI121-antiBnCCoAOMT)和cDNA全长表达质粒(pWM101-BnCCoAOMT),并通过根癌农杆菌介导法将其转化至模式烟草WS38,获得了转基因烟草.对转基因植株进行分子分析和组织学初步研究表明,转反义RNA基因植株叶柄木质素含量较野生烟草或转正义基因烟草叶柄木质素含量降低.说明运用反义RNA技术对CCoAOMT基因的表达进行基因工程调控,一定程度上可以对木质素的合成产生干扰,为获得低木质素或木质素组分改良的苎麻基因工程奠定基础.     

木质素的生物合成及其调控研究进展

木质素是植物体中仅次于纤维素的一种重要大分子有机物质,具有重要生物学功能,其3种主要单体的生物合成途径已经基本清楚。从木质素生物合成及基因工程在调控木质素生物合成中的作用等方面的研究进展进行了综述,并提出了存在的问题及对策。     

3种木质素的主要理化性质分析

介绍了木质素、碱木质素和铵化木质素的制备方法,且对这3种木质素的比重、钠元素含量、X射线衍射、热重、溶解度等理化性质进行表征。研究结果表明,铵化木质素不含有碱金属钠,同时具有很好的水溶性,即铵化木质素解决了纯木质素难溶于水的问题,又解决了碱木质素与柴油乳化后对柴油发动机汽缸内的损坏和长期使用会存在积炭的风险。这说明铵化木质素与木质素和碱木质素相比更适于同柴油乳化混合,实现传统化石能源的添加剂,铵化木质素为我国林木废弃生物质资源化利用和替代能源开发提供了一条潜在的途径。     

Role of oxidative enzymatic treatments on enzymatic hydrolysis of softwood

The impact of oxidative modification and partial removal of lignin by laccase-mediator treatments on the enzymatic hydrolysis of steam-pretreated softwood (SPS) was evaluated. Two mediators, N-hydroxy-N-phenylacetamide (NHA) and its acetylated precursor, were oxidized by the laccase from Trametes hirsuta, and their effects on the activity of cellulolytic enzymes and on the hydrolysis yield of SPS were examined. Both simultaneous and sequential combinations of laccase-mediator treatments with commercial cellulases increased the sugar yield in the enzymatic hydrolysis of SPS. The maximal increase was 21% when a sequential treatment was applied. Laccase treatment alone was also shown to improve hydrolysis. NHA oxidized by laccase inhibited significantly the cellulases of Trichoderma reesei, but the presence of the solid substrate protected the activities against oxidative inactivation. Surface analysis of the lignocellulosic substrate before and after the laccase and cellulase treatments revealed an enrichment of lignin and an increase of carboxylic groups on the surface of the hydrolysis residue.     

Distribution and roles of p-hydroxycinnamate: CoA ligase in lignin biosynthesis

p-Hydroxycinnamate:CoA ligases were extracted from the xylems of angiosperms and gymnosperms, and the substrate specificities toward ferulate and sinapate were examined. Most of angiosperm and gymnosperm CoA ligases examined were active with ferulate but not with sinapate; however, the enzymes of Erythrina crista-galli, Robinia pseudoacacia and bamboo showed considerable activity with sinapate. The other enzymes, although inactive with sinapate, showed no inhibitory effect on the Erythrina CoA ligase reaction with sinapate. The K_ms for sinapate and ferulate of the Erythrina enzyme were 1.0 and 2.1 μM, respectively, and p-hydroxycinnamate was the best substrate among cinnamates examined. The MW of the CoA ligase was 40 000 and the pH optimum was between 7.2 and 7.6. The possible roles of p-hydroxycinnamate:CoA ligase in lignin biosynthesis are discussed.     

木质素柴油制备工艺优化与理化性质评价

介绍了一种新型木质素柴油的制备工艺优化方法及其理化性质评价。根据添加木质素的比例、乳化剂的添加量和等影响因素进行正交试验设计,分别选择添加木质素的量为总质量2%、3%和4%的比例,以乳化剂的用量为4%、5%和6%,HLB为8.3、8.8和9.3以及0.2、0.3和0.4 g的助乳化剂用量的不同等条件为水平标准,配制了多种不同比例的乳化油,最终筛选乳化条件为：HLB值为9.3,乳化剂的用量为6.0%,助乳化剂的加入量为1.2%,乳化温度为室温,乳化时间2 min。同时还针对氧化安定性、含硫量、酸度、10%蒸余物残炭、铜片腐蚀、机械杂质、运动粘度、凝点、闪点（闭合）、十六烷值、馏程、密度十二项指标进行了国家标准检测均高于GB 252-2000轻柴油的国家标准。其中木质素柴油的十六烷值均到了46.1和48.8,远远超过了45的国家标准;这对开发柴油的替代燃料,缓解燃料供需紧张矛盾,降低燃料消耗率等都有重大意义。     

Biosynthesis and Genetic Engineering of Lignin

Lignin, a complex heteropolymer of cinnamyl alcohols, is, second to cellulose, the most abundant biopolymer on Earth. Lignification has played a determining role in the adaptation of plants to terrestrial life. As all extracellular polymers, lignin confers rheological properties to plant tissues and participates probably in many other functions in cell and tissue physiology orin cell-to-cell communication. Economically, lignin is very important because it determines wood quality and it affects the pulp and paper-making processes as well as the digestibility of forage crops. For all these reasons the lignin biosynthesis pathway has been the subject of many studies. At present, most genes encoding the enzymes involved in the biosynthesis of lignin have been cloned and characterized. Various recent studies report on the alteration of the expression of these genes by genetic engineering, yielding plants with modified lignin. In addition, several mutants have been analyzed with changes in lignin content or lignin composition resulting in altered properties. Thanks to these studies, progress in the knowledge of the lignin biosynthesis pathway has been obtained. It is now clear that the pathway is more complex than initially thought and there is evidence for alternative pathways. A fine manipulation of the lignin content and/or composition in plants is now achievable and could have important economical and environmental benefits.     

Downregulation of caffeoyl-CoA O-methyltransferase (CCoAOMT) by RNA interference leads to reduced lignin production in maize straw

Lignin is a major cell wall component of vascular plants that provides mechanical strength and hydrophobicity to vascular vessels. However, the presence of lignin limits the effective use of crop straw in many agroindustrial processes. Here, we generated transgenic maize plants in which the expression of a lignin biosynthetic gene encoding CCoAOMT, a key enzyme involved in the lignin biosynthesis pathway was downregulated by RNA interference (RNAi). RNAi of CCoAOMT led to significantly downregulated expression of this gene in transgenic maize compared with WT plants. These transgenic plants exhibited a 22.4% decrease in Klason lignin content and a 23.3% increase in cellulose content compared with WT plants, which may reflect compensatory regulation of lignin and cellulose deposition. We also measured the lignin monomer composition of the RNAi plants by GC-MS and determined that transgenic plants had a 57.08% higher S/G ratio than WT plants. In addition, histological staining of lignin with Wiesner reagent produced slightly more coloration in the xylem and sclerenchyma than WT plants. These results provide a foundation for breeding maize with low-lignin content and reveal novel insights about lignin regulation via genetic manipulation of CCoAOMT expression.     

The influence of lignin migration and relocation during steam pretreatment on the enzymatic hydrolysis of softwood and corn stover biomass substrates

To be effective, steam pretreatment is typically carried out at temperatures/pressures above the glass transition point (Tg) of biomass lignin so that it can partly fluidize and relocate. The relocation of Douglas-fir and corn stover derived lignin was compared with the expectation that, with the corn stover lignin's lower hydrophobicity and molecular weight, it would be more readily fluidized. It was apparent that the Tg of lignin decreased as the moisture increased, with the easier access of steam to the corn stover lignin promoting its plasticization. Although the softwood lignin was more recalcitrant, when it was incorporated onto filter paper, it too could be plasticized, with its relocation enhancing enzymatic hydrolysis. When lignin recondensation was minimized, the increased hydrophobicity suppressed lignin relocation. It was apparent that differences in the accessibility of the lignin present in Douglas-fir and corn stover to steam significantly impacted lignin fluidization, relocation, and subsequent cellulose hydrolysis.     

The effect of fungal laccase on fractionated lignosulphonates (peritan Na)

Two fractions obtained after chromatography of lignosulphonates on Sephadex G-50, varying in M_rs, were treated with extracellular Trametes versicolor laccase. After incubation of the low M_r fraction, polymerization was observed, while in the case of the high M_r fraction the reverse process occurred. As a result of depolymerization, five new lower M_r fractions appeared. The reaction reached peak level after 2 hr of incubation and then the quantities of the products diminished, possibly due to their repolymerization. These studies indicate that laccase possesses both polymerization and depolymerization activity though the former was predominant.     

木质素生物合成途径及调控的研究进展

木质素是植物体中仅次于纤维素的一种重要大分子有机物质,具重要生物学功能。木质素填充于纤维素构架中增强植物体的机械强度,利于疏导组织的水分运输和抵抗不良外界环境的侵袭。陆生植物的木质素合成是适应陆地环境的重要进化特征之一。然而,制浆造纸的中心环节是用大量化学品将原料中的木质素与纤维素分离,纤维素用于造纸,分离的木质素等成为造纸工业的主要废弃物,对江河湖海的污染触目惊心。脱木质素的化学品投入及废液的碱回收处理需大量耗能并增加造纸成本。饲草的木质素还影响牲畜的消化与营养吸收,木质素含量的高低是饲草优劣…