木质素合成关键酶基因与造纸植物转基因改良应用研究

木质素是自然界数量仅次于纤维素的自然有机物质,但在造纸业中是主要的污染来源。木质素生物合成过程十分复杂,涉及了大量酶体系。综述了木质素合成途径中的关键调控酶特性及其基因调控情况,从培育低木质素、易降解木质素的造纸植物为出发点,分析了这些关键基因在转基因造纸植物应用中的潜力和存在的主要问题,并提出了应从改变植物体内木质素含量、改变木质素单体构成比例这两个方面进行转基因的研究思路。     

RNA干涉培育低木质素杨树

采用改良的CTAB法提取南林95杨的基因组DNA,经PCR扩增得到肉桂酰辅酶A还原酶CCR基因的第4个外显子部分序列,通过中间载体pUCCRNAi,构建含正反向干涉片段的pBI121表达载体,导入农杆菌LBA4404。利用叶盘法侵染南林95杨,获得3株转基因植株,经分子鉴定证实干涉片段已导入南林95杨。测定Klason木质素及综纤维素含量的结果显示:转基因植株Klason木质素含量与对照相比平均降低了9.86%,综纤维素含量与对照相比平均增加了3.17%,纤维长宽比明显增加,均表明转基因植株更有利于造纸。     

RNA干涉培育低木质素杨树

采用改良的CTAB法提取南林95杨的基因组DNA，经PCR扩增得到肉桂酰辅酶A还原酶CCR基因的第4个外显子部分序列，通过中间载体pUCCRNAi，构建含正反向干涉片段的pBll21表达载体，导入农杆菌LBA4404。利用叶盘法侵染南林95杨，获得3株转基因植株，经分子鉴定证实干涉片段已导入南林95杨。测定Klason木质素及综纤维素含量的结果显示：转基因植株Klason木质素含量与对照相比平均降低了9．86％，综纤维素含量与对照相比平均增加了3．17％，纤维长宽比明显增加，均表明转基因植株更有利于造纸。     

橡胶树HbMYB20基因的克隆及其对拟南芥次生壁发育的调控

【目的】MYB转录因子是调控植物木质素合成和次生壁形成的重要转录因子之一。本文分离克隆到一个与拟南芥AtMYB20高度同源的橡胶树MYB转录因子基因HbMYB20,并在拟南芥中对其功能进行研究,以期了解其在橡胶树木质素合成和次生壁发育的分子调控中的作用,为橡胶树木材形成的分子调控机制研究及其遗传改良奠定基础。【方法】采用 blast分析从树皮转录组中筛选出与拟南芥 AtMYB20序列同一性较高的橡胶树 MYB 基因HbMYB20;设计 ORF区特异性引物,以树皮 cDNA 为模板进行扩增得到该目的基因 cDNA 序列。实时荧光定量PCR检测该基因在橡胶树叶片、胶乳、茎干以及木质部与韧皮部的相对表达量。构建 HbMYB20过表达植物载体,使用农杆菌蘸花法转化拟南芥,获得该基因过表达转基因株系。采用乙酰溴法和间苯三酚染色法,分析转基因、野生型拟南芥茎的木质素含量以及木质素在拟南芥茎基部横截面中的分布。对转基因、野生型拟南芥茎基部横截面切片进行甲苯胺蓝染色,并测量分析导管、木质纤维和维管束间纤维细胞的细胞壁厚度。最后,采用实时荧光定量PCR分析转基因及野生型拟南芥木质素和纤维素合成相关酶基因的表达。【结果】克隆得到1个橡胶树 MYB 转录因子基因 HbMYB20,该基因开放阅读框( ORF)为927 bp,编码309aa 的蛋白,氨基酸序列分析显示,HbMYB20与AtMYB20/43和 AtMYB85/42同源性较高,属 R2R3MYB转录因子 G8亚组成员。表达分析显示 HbMYB20在橡胶树茎干和木质部中高表达,胶乳中表达最低。对 HbMYB20过表达拟南芥分析显示,该基因在3个转基因株系中均表达;相对野生型拟南芥,转 HbMYB20拟南芥植株生长抑制,木质部和维管束间纤维的木质素染色面积较少、染色程度变浅,茎的木质素含量和木质纤维、导管及维管束间纤维的细胞壁厚度均显著低于野生型;同时转基因株系中木质素合成关键酶基因4CL1和 CCoAOMT的表达量以及纤维素合成关键酶基因 CesA8的表达显著下调。【结论】橡胶树 MYB转录因子 G8亚组成员 HbMYB20,在茎和木质细胞中高表达。拟南芥中过表达 HbMYB20导致转基因植株的矮小,细胞壁变薄,阻碍木质部中木质素的合成和积累,同时木质素和纤维素合成相关酶基因的表达显著下降。由此推测 HbMYB20对拟南芥的木质素和纤维素合成都具有负调控作用,可能是1个橡胶树次生壁发育的负调控因子。     

基因工程对林木生长表型与细胞壁组分及构造的影响研究

通过基因工程技术培养出木质素含量低、纤维素含量高和糖转化效率高以及优质的木材,对于将其定向应用于制浆造纸、生物炼制、木质建筑及装饰材料方面具有重要的研究意义。文章详细阐明了木质素和纤维素基因调控技术对转基因林木生长表型、细胞壁化学组分含量及其微区分布、组织细胞形态及细胞壁超微构造影响的研究进展,并对转基因林木今后的重点发展方向进行了展望,以期为我国定向培育优质速生人工林提供理论依据。     

通过RNAi技术抑制杨树c3h基因表达提高糖转化效率

利用克隆得到的毛白杨c3h1基因构建其RNAi抑制表达载体,通过根癌农杆菌介导的叶盘法转化银腺杨无性系84 K,Realtime PCR检测表明其转基因株系323、325和322中c3h1基因表达量较野生型植株分别下调89.04%、82.22%和68.38%;茎横切片组化染色和显微结构观察表明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、纤维素含量测定及苯酚—硫酸法总糖含量与HPLC法可溶性总糖和单糖含量检测结果表明:转基因植株木质素含量平均降低23.00%,最高可达39.71%;酸前处理效率最高提高了41.39%;未经酸处理直接酶解的糖化效率是对照植株的2.34～2.72倍,322株系和323株系比对照植株经酸前处理后再酶解的糖化效率高出81.18%和375.53%。     

离体毛竹笋纤维素和木质素含量及POD和PAL活性研究

研究了离体后毛竹笋纤维素、木质素含量以及苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)活性的变化.结果显示:笋体纤维素、木质素含量从笋尖到笋基部逐渐增加;PAL、POD活性笋尖明显高于笋基部.离体后贮藏的竹笋纤维素、木质素含量随贮藏时间的延长而增加,在前5 d纤维素的增加速度极快,在后5 d增加速度明显趋缓,但木质素含量随贮藏时间的延长基本呈匀速态势增加;POD、PAL活性随贮藏时间的延长,活性均显著增加.对离体竹笋进行低温处理(4 ℃)可显著降低竹笋的PAL、POD活性,减少木质素和纤维素合成,低温对纤维素的影响高于木质素.     

杉木、I-72杨主要化学组成的株内纵向变异研究

对杉木和I-72杨的心、边材的主要化学组成沿树高方向的株内纵向变异进行了研究。主要研究内容为综纤维素、α-纤维素、半纤维素、木质素、酸溶木质素的含量及综纤维素与木质素的比值变化等。研究结果揭示了这两个树种的心、边材的主要化学成分在纵向上的变异规律：杉木和I-72杨的心、边材综纤维素和α-纤维素的含量均为边材高于心材,但在沿树高方向的纵向变化规律各异;杉木、I-72杨心、边材的半纤维素含量沿着树高方向表现出不同的变化规律;杉木木质素在各个树高部位均为边材高于心材,边材木质素含量随着树高的增加逐步降低,而心材木质素含量则沿树高方向先增加然后逐步降低。I-72杨心、边材木质素含量的变化表现各异,心材含量随着树高的增加逐步增加,边材则表现为无规律变化;杉木、I-72杨心、边材的酸溶木质素含量均沿树高方向逐渐降低,但杉木边材的酸溶木质素含量高于心材,而I-72杨却是心材高于边材;综纤维素与木质素的比值在沿树高方向具有不同的纵向变化规律。     

木质素生物合成及其基因调控的研究进展

木质素是地球上数量仅次于纤维素的有机物, 在植物生长发育中具有重要的生物学功能, 也是生物质能源的来源之一, 但在制浆造纸过程中, 将木材原料中木质素与纤维素分离, 不仅能耗高, 成本高, 而且废弃物还污染环境.林木木质素改良对于提高纸浆得率和质量、降低造纸经济成本以及环境保护, 都具有重要意义.文中介绍了木质素生物合成途径及其基因调控的研究进展, 此外, 还介绍了木质素生物合成基因调控的研究趋势, 主要是木质素特异性启动子、双价和多基因结构的共抑制以及转录因子的调控.     

杨树木质素合成基因C3H与HCT表达下调对细胞壁空间形态的影响

木质纤维素是地球上数量最大的可再生资源,由木质素、半纤维素及纤维素三者紧密结合产生的抗降解屏障作用是纤维素能源利用的主要障碍。高效分离木质素,须对木质素在木材细胞壁中的结构与分布进行充分的研究。本研究中选定莽草酸/奎尼酸羟基肉桂酰转移酶HCT基因和毛白杨香豆酰莽草酸/奎宁酸羟化酶C3H基因为调控目标,通过根癌农杆菌叶盘转化法转化银腺杨无性系84K,最终得到C3H-RNAi与HCT-RNAi转基因植株。发现C3H-RNAi和HCTRNAi转基因株系比野生型植株内中外三层木质部的平均导管腔茎、导管壁、木纤维腔径、木纤维壁均变小。壁腔比值都小于1,比较适合作为造纸纤维原料。探索通过基因改良技术改变杨树木质素结构在细胞壁中空间尺度上分布变化,这将为林木材性改良,木质纤维素高效利用研究奠定基础。     

Profiling of phenylpropanoid monomers in developing xylem tissue of transgenic aspen (Populus tremuloides)

Here we describe alterations in the cinnamate/monolignol pathway in three transgenic aspen lines: one with downregulated expression of 4-coumarate:CoA ligase (4CL), one with upregulated expression of coniferaldehyde 5-hydroxylase (CAld5H), and a 4CL downregulated/CAld5H upregulated line. Compared with the wild type, the 4CL downregulated line showed significantly increased levels of p-hydroxycinnamic acids such as p-coumaric, ferulic, and sinapic acids. In contrast, the CAld5H upregulated line had increased content of p-coumaryl and 5-hydroxyconiferyl alcohols. In the 4CL downregulated line, it was likely that most hydroxycinnamic acids were glycosylated. These results strongly suggest that the downregulation of 4CL and upregulation of CAld5H disrupt the metabolic flow through the cinnamate/monolignol pathway and thus alter the amount and structure of its final product, lignin.     

Multivariate modeling of acoustomechanical response of 14-year-old suppressed loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) to variation in wood chemistry,microfibril angle and density

The polymeric angle and concentration within the S₂ layer of the softwood fiber cell wall are very critical for molecular and microscopic properties that influence strength, stiffness and acoustic velocity of wood at the macroscopic level. The main objective of this study was to elucidate the effect of cellulose, hemicellulose, lignin, microfibril angle and density on acoustic velocity and material mechanical properties of 14-year-old suppressed loblolly pine. Cellulose, hemicellulose and density are consistently the most important drivers of strength, stiffness and velocity. Cellulose and lignin are the highest and lowest contributor to velocity, respectively, with lignin acting as a sound wave dispersant, while cellulose is the most important conductor of sound wave at the molecular level, while hemicellulose acts as a special coupling agent between these components. The polymeric constituents are thus important drivers of sound wave propagation at the molecular level, while density played a subsequent role at the macroscale.     

PCR法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA

4 香豆酸 :CoA连接酶 (4CL)是木质素生物合成过程中重要的酶 .该文利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法直接获得了紫穗槐 4CLAcDNA全长序列 ,避免了复杂的构建、筛选cDNA文库的过程 .先用简并PCR得到了 4CLA1片段 ,又据此片段设计反向嵌套引物 ,用RACE方法获得未知的 5′和 3′端序列 .所获得的全长 4CLAcDNA ,编码 5 40个氨基酸 .氨基酸序列同源性分析表明4CLA是典型的 4CL蛋白 ,含有预计的AMP binding位点、催化反应区和保守的Cys .     

Cloning and analysis of a new 4CL-like gene in Populus tomentosa

The phenylpropanoid enzyme 4-coumarate: coenzyme A ligase (4CL), plays a key role in general phenylpropanoid metabolism. Our investigation involves a new 4CL-like gene cloned from Populus tomentosa. This new 4CL-like gene is 1,692 bp and encodes a protein of 552 aa. After analysis of its domain, we found a highly conserved region of a 4CL gene family in this 4CL-like gene. Based on our results, we believe that this gene belongs to the family of 4CL. A recombinant vector, referred to as pET30a-4CL-like, was constructed by connecting this 4CL-like gene fragment to pET30a. After inducing it with IPTG for 3 h, the 4CL-like protein was purified by a Ni-NTA method. This 4CL-like protein has a calculated molecular weight of 60 kDa by SDS-PAGE.     

Recrystallization behavior of cellulose and lignocellulose from Pinus massoniana

Summary In an X-ray diffraction investigation, the recrystallization behavior of ball-milled Chinese southern pine wood was found to differ from that of cellulose. The crystallinity of lignocellulose milled for a very long time and recrystallized by wetting in water did not decrease with increasing milling time as was the case with milled cellulose. In addition, it appeared that the presence of lignin tended to restrict the amorphous cellulose produced by milling from recrystallizing into Cellulose II, whereas it had no influence on the recrystallization into Cellulose I. These results were discussed in the context of the mechanism of the conversion of Cellulose I to Cellulose II.     

Radiation-use efficiency of a forest exposed to elevated concentrations of atmospheric carbon dioxide

We compared radiation-use efficiency of growth (epsilon;), defined as rate of biomass accumulation per unit of absorbed photosynthetically active radiation, of forest plots exposed to ambient (approximately 360 micro l l-1) or elevated (approximately 560 micro l l-1) atmospheric CO2 concentration ([CO2]). Large plots (30-m diameter) in a loblolly pine (Pinus taeda L.) plantation, which contained several hardwood species in the understory, were fumigated with a free-air CO2 enrichment system. Biomass accumulation of the dominant loblolly pines was calculated from monthly measurements of tree growth and site-specific allometric equations. Depending on the species, leaf area index (L*) was estimated by three methods: optical, allometric and litterfall. Based on the relationship between tree height and diameter during the first 3 years of exposure, we conclude that elevated [CO2] did not alter the pattern of aboveground biomass allocation in loblolly pine. There was considerable variation in L* estimates by the different methods; total L* was 18-42% lower when estimated by the optical method compared with estimates from allometric calculations, and this discrepancy was reduced when optical measurements were corrected for the non-random distribution of loblolly pine foliage. The allometric + litterfall approach revealed a seasonal maximum total L* of 6.2-7.1 with about 1/3 of the total from hardwood foliage. Elevated [CO2] had only a slight effect on L* in the first 3 years of this study. Mean epsilon; (+/- SD), calculated for loblolly pine only, was 0.49 +/- 0.05 and 0.62 +/- 0.04 g MJ-1 for trees in the ambient and elevated [CO2] plots, respectively. The 27% increase in epsilon; in response to CO2 enrichment was caused primarily by the stimulation of biomass increment, as there was only a small effect of elevated [CO2] on L* during the initial years of fumigation. Long-term increases in atmospheric [CO2] can increase epsilon; in closed-canopy forests but the absolute magnitude and duration of this increase remain uncertain.