利用拟南芥基因芯片和突变体对木材形成相关基因的初步分析

木材形成是木本植物特有的生物学过程,拟南芥在适当的诱导条件下也能形成类似“木材”的维管组织,因而可以借助拟南芥丰富的基因资源研究木材形成的分子机制.利用前期建立的毛白杨次生维管系统再生实验体系,通过拟南芥表达谱芯片分析再生过程中的基因表达变化,获得149个差异表达基因.选择其中转录因子等调控基因及功能未知基因共89个基因的总计151个拟南芥突变体,经次生诱导培养发现,20个突变体的发芽率或成活率降低,10个突变体表型变化明显,出现维管系统次生生长发育受到抑制、生长速度减慢等,推测这些基因参与调控拟南芥的次生生长.将木本植物与草本植物的研究体系相结合,利用拟南芥次生生长诱导体系研究木材发育相关基因功能,为木材发育的基因功能研究提供一条可行、有效,快速解析基因功能的新途径.     

烟草和毛白杨次生维管发育相关MYB转录因子分析

[目的]为探讨额河杨和银灰杨天然杂种的起源机制,[方法]应用18对SSR标记,从分子水平上对新疆额尔齐斯河流域杨属植物的种间关系进行分析研究。[结果]表明:(1)SSR系统发育树将整个流域天然杨属植物分为两大类群,即黑杨派和青杨派为一类,白杨派为一类;(2)白杨派派内系统聚类图显示,银白杨、欧洲山杨、银灰杨三个树种均有较大的遗传分化,特别是杂种银灰杨似乎更大;(3)黑杨派和青杨派的UPGMA分类图显示,青杨派和黑杨派分属于2个分支,其中,青杨派内部分化相对简单,分为2支,均为典型的苦杨;黑杨派内部的分化较为复杂,可分为4类,包括典型的欧洲黑杨、额河杨和回交子代。[结论]杂种额河杨具有更多的欧洲黑杨的遗传成分,因此,将额河杨放到黑杨派是正确的。     

杨树BAG基因的鉴定及表达模式分析

【目的】杨树是我国人工林的重要组成部分,也是公认的模式木本植物。植物BAG蛋白作为保守的分子伴侣,在生长发育和抗逆性中起到关键作用。研究杨树BAG蛋白家族的进化与表达模式,鉴定BAG基因的生物学功能对于林木遗传改良具有重要的指导意义。【方法】以拟南芥7个BAG蛋白为诱饵,搜索毛果杨、水稻和小立碗藓在线数据库(https://phytozome.jgi.doe.gov)获得这3个物种同源的BAG蛋白。利用生物信息学构建毛果杨、拟南芥、水稻和小立碗藓BAG蛋白的系统进化树,分析这些蛋白的生化特性及进化关系。结合杨树芯片(http://bar.utoronto.ca)数据,利用qRT-PCR检测杨树BAG基因的组织和逆境(旱和热胁迫)表达模式。【结果】毛果杨包含14个BAG基因,按照国际命名法(以染色体位置进行排序),将其命名为PtrBAG1—PtrBAG14。系统进化树表明杨树、拟南芥、水稻和小立碗藓BAG蛋白相对保守,大体可以分为2个亚家族。第1个亚家族中,大部分BAG蛋白N端含有UBL结构域,可能作为分子桥梁参与降解某些蛋白;第2个亚家族的大部分BAG蛋白含有IQ结构域,意味着这些蛋白可能与Ca²⁺信号相关。杨树BAG基因在不同逆境处理(热胁迫和干旱胁迫)条件下有不同的表达模式。热处理诱导所有BAGs基因表达发生变化,其中BAG1、BAG4、BAG6、BAG7和BAG8表达被激活,而另9个BAG基因的表达被抑制;特别是,热诱导BAG4和BAG6表达上调超过90倍。干旱胁迫条件下,大部分BAG表达变化幅度很小。组织表达模式分析表明,大部分杨树BAG基因在根、叶、芽、小苗、雌花序、雄花序和木质部中表达量很低,只有BAG2和BAG12在木质部中大量表达。【结论】杨树BAG家族共有14个成员,大体分为2个亚家族,在进化上与本文选用的陆生植物拟南芥和水稻BAG蛋白有更高的相似性。杨树14个BAG基因可能都参与了热胁迫调控,但不同成员在抗热过程中起不同作用。相比而言,大部分杨树BAG基因可能并不参与抗旱。值得指出的是,杨树BAG4和BAG6可能在热胁迫中作用最明显,而BAG2和BAG12可能参与木材形成。     

Seasonal changes in gene expression at the sapwood-heartwood transition zone of black locust (Robinia pseudoacacia) revealed by cDNA microarray analysis

Heartwood is a determining factor of wood quality and understanding the biology of heartwood may allow us to control its formation. Heartwood formation is a form of senescence that is accompanied by a variety of metabolic alterations in ray parenchyma cells at the sapwood-heartwood transition zone. Although senescence has been studied at the molecular level with respect to primary growth, the cell maturation and death events occurring during heartwood formation have been difficult to study because of their location and timing. Analysis of global gene expression patterns during the transition from sapwood to heartwood may offer a powerful means of identifying the mechanisms controlling heartwood formation. Previously, we developed cDNA microarrays carrying 2567 unigenes derived from the bark/cambium region, sapwood and transition zone of a mature black locust tree. Here, we describe the use of these microarrays to characterize seasonal changes in the expression patterns of 1873 genes from the transition zone of mature black locust trees. When samples collected in summer and fall were compared, 569 genes showed differential expression patterns: 293 genes were up-regulated (> twofold) in summer (July 5) and 276 genes were up-regulated in fall (November 27). More than 50% of the secondary and hormone metabolism-related genes on the microarrays were up-regulated in summer. Twenty-nine out of 55 genes involved in signal transduction were differentially regulated, suggesting that the ray parenchyma cells located in the innermost part of the trunk wood react to seasonal changes. We established the expression patterns of 349 novel genes (previously unknown or no-hit), of which 154 were up-regulated in summer and 195 were up-regulated in the fall.     

拟南芥MYB基因对次生维管系统发育的影响

根据拟南芥ATH1全基因芯片分析毛白杨维管再生过程中基因表达谱的结果,选取了在芯片中特异表达且表达量较高的MYB转录因子家族的7个基因,通过拟南芥突变体研究其对次生维管系统发育的影响及与木材形成相关的关系。通过"三引物法"的纯杂合鉴定和切片显微观察,发现其中1个突变系只有纯合突变体没有杂合体,并且茎基部维管束间纤维细胞壁和野生型相比明显增厚,细胞数量减少,细胞腔也随之减小,推测该突变体可能与次生壁的沉积有关系。     

巨桉EXP基因家族的生物信息学分析

[目的]本研究有助于了解EXP基因家族的基本特征,为深入研究其功能搭建平台。[方法]本研究对从巨桉(Eucalyptus grandis Hill)中筛选出35个EXP基因家族成员(Egr EXP1 Egr EXP35),利用生物信息学方法对其基因特征与表达模式进行综合分析。[结果]巨桉EXP基因分布在8条染色体之上,EXP蛋白均定位在细胞质膜上发挥作用,大多数的家族成员具有信号肽。巨桉EXP编码的蛋白质由α-螺旋、延伸链、无规卷曲、β-转角组成。进化分析结果表明,巨桉EXP蛋白与毛果杨(Populus trichocarpa) EXP蛋白的进化关系接近。35个巨桉EXP基因在巨桉未成熟木质部、成熟叶片、韧皮部、茎尖、木质部以及幼叶组织中表达模式存在显著差异。[结论]EXP基因家族各成员的表达模式不同,Egr EXP17、Egr EXP18可能在巨桉木材形成过程中起重要作用。     

杨木和辐射松树脂增强改性材机械加工性能研究

为考察杨木和辐射松树脂浸渍材的机械加工性能,参照LY/T 2054—2012《锯材机械加工性能评价方法》,分别对杨木和辐射松的素材与树脂浸渍材及其经过真空热处理的树脂浸渍材的刨削、铣削、钻削、开榫和车削5种机械加工性能进行测试评价。结果表明:1)本试验范围内,在综合评分满分40分情况下,杨木素材、树脂浸渍材和真空热处理树脂浸渍材的总评分分别为20分、30分和25分;辐射松素材、树脂浸渍材和真空热处理树脂浸渍材的总评分分别为24分、33分和29分;树脂浸渍材机械加工综合性能最佳,热处理树脂浸渍材次之;2)杨木和辐射松的树脂浸渍材及其真空热处理树脂浸渍材的刨削、铣削性能均为优秀,车削性能均为良好;杨木和辐射松的树脂浸渍材及辐射松真空热处理树脂材开榫性能良好;钻削性能仅辐射松树脂浸渍材达到优秀;3)辐射松素材与改性处理材的机械加工性能均优于杨木,综合比较,辐射松树脂浸渍材具有较佳的机械加工性能。     

Differential expression of genes encoding cell wall proteins in vascular tissues from vertical and bent loblolly pine trees

Differential expression of cell wall proteins during plant development and in response to biotic or abiotic stress suggests that these proteins may contribute in different ways to plant cell wall architecture. Because the wood of loblolly pine (Pinus taeda L.) is highly specialized in the formation of secondary cell walls, it is an ideal tissue for studying these proteins. The cDNAs coding for six novel cell wall associated proteins, as well as a homologue for a phytocyanin, were identified and characterized from differentiating xylem of loblolly pine. Three of these cDNAs encoded new putative loblolly pine arabinogalactan proteins, based on their structural similarity to classical arabinogalactan proteins (AGPs). In addition, one clone was related to the proline-rich protein group and the other two to the glycine-rich protein group and the mussel adhesive protein. Relative expression of these genes was examined in different tissues and organs from normal trees (needles, phloem and vertical wood), the underside of bent trees (compression wood) and the lateral sides of the bent stems (side wood). All clones, except one, were highly expressed in vascular tissues with noticeable differences among the three types of wood. Their relationships and diversity provide the first insights into concerted expression and related function for this important group of proteins in cell wall formation of wood.     

Seasonal variation in gene expression for loblolly pines (Pinus taeda) from different geographical regions

In developing xylem, gene expression levels vary in different genotypes, at different stages of development, throughout a growing season, and in response to stresses. Commercially important characteristics such as wood-specific gravity are known to differ with seed source. For example, when grown on a common site, the specific gravity of Arkansas loblolly pine (Pinus taeda L.) trees is greater than that of Louisiana loblolly pine, and Texas loblolly pines have a greater specific gravity than loblolly pines from the Atlantic coast. A microarray analysis was performed to examine variation in gene expression among trees from different geographical sources when grown on a common site, and seasonal variation in gene expression in each seed source. We used microarrays containing 2171 expressed sequence tags (ESTs) with putative functions of interest, selected from several loblolly pine xylem partial cDNA libraries and a shoot tip library. Genes with significant variation in expression for each factor were identified. Many genes preferentially expressed in latewood compared with earlywood were for proteins involved in cell wall biosynthesis. Variation in gene expression among trees from the two seed sources in each growing season suggests that there may be more differences between South Arkansas trees and South Louisiana trees in latewood than in earlywood. Variation in gene expression among trees from different regions may reflect adaptation to different environments.     

Occurrence and significance of bacteria in living trees of Populus nigra L.

On the occurrence and significance of bacteria in living trees of Populus nigra L. Eleven strains of bacteria were isolated from sapwood and heartwood of living poplar trees (Populus nigra L.) and identified mostly as Erwinia, Xanthomonas, Agrobacterium and Acinetobacter. Most of them were able to attack milled wood and the wood components pectin, hemicelluloses and holocellulose; α-cellulose and lignin were not consumed. The capillary liquid in the xylem of poplar served as a nutrient for the isolated bacteria. The significance of these bacteria for wetwood formation is discussed.     

杨树油菜素内酯合成基因DET2的克隆与功能分析

[目的]DET2基因编码一个5α-还原酶,是油菜素内酯(BRs)合成过程中的关键限速基因。研究DET2基因在杨树生长发育中的作用,对于进一步研究油菜素内酯在木本植物中的调控机制有重要意义。[方法]从银腺杨84K(Populus alba×P. glandulosa,‘84K’)克隆得到拟南芥AtDET2同源基因PagDET2,利用生物信息学对其进行序列比对、生化特征分析、构建系统发育进化树。通过RT-PCR分析其在杨树中的表达模式。构建由CaMV 35S强启动子驱动的过表达载体,通过农杆菌介导的叶盘转化法转化84K杨,得到PagDET2-OE转基因植株。分析过表达DET2基因对于转基因植株内源BRs含量、植株生长和抗逆的影响。[结果]克隆了包含全长编码区PagDET2基因全长,可编码一个长度为257个氨基酸的蛋白质。其蛋白序列与毛果杨、拟南芥、水稻、棉花、大豆、番茄DET2蛋白同源性较高,说明该基因在进化过程中相对保守。PagDET2在84K杨不同组织中均检测到表达,其在茎中的表达较高。通过ELISA检测植物BRs含量发现,过量表达DET2可以显著提高杨树内源BRs含量。过量表达DET2基因,可以导致转基因植株的高生长,但对盐胁迫更加敏感。[结论]DET2基因作为BRs合成的关键基因,在杨树中过量表达可以显著提高内源BRs含量,促进植株增高。DET2转基因植株的获得为进一步分析BR参与木本植物生长发育的调控机制奠定了基础。     

A database for poplar gene co-expression analysis for systematic understanding of biological processes,including stress responses

Reforestation in the humid tropics and arid zones, where trees are often subject to stresses, is an effective approach for mitigating global warming. Forestation with Populus species that are tolerant to the stresses in such regions has been conducted. The selection of poplar trees with higher stress tolerance leads to more efficient reforestation. The genome-wide bioinformatics approaches of gene function have been used for revealing the mechanisms of biological processes, including such stress tolerance. The decoding of the poplar genome has been followed by the genome-wide identification of genes and then the inference of gene function for systematic understanding of biological processes. To predict gene function in poplar, we analyzed poplar gene expression data using DNA microarray datasets obtained from the Gene Expression Omnibus database and developed a database for poplar gene co-expression analysis. Using the database, we illustrate the steps to retrieve two groups of co-expressed genes that are specifically expressed in experiments of hypoxic stress response in gray poplar, a flooding-tolerant tree species. Our database allows users to extract genes involved in biological processes, such as stress reaction, and then is useful for understanding such mechanisms in tree species.     

北美地区杨树木材利用现状与前景

过去20多年, 北美地区的杨树已经从杂草树种转变为产生巨大经济效益的用材林树种, 杨树木材因其具有较高纤维素含量和较低的木质素含量, 适合用作纸浆和纸制品的原料。杨木制浆方法有机械法、化学法、半化学法和化学机械法, 杨木浆可以作为高级纸、纸板、新闻纸和包装纸。杨木牛皮纸浆与针叶材牛皮纸浆混合, 具有良好的成纸性能, 透明度高, 适合制作高级用纸。杨木也是传统的锯材、单板和胶合板工业的原材料, 用来生产锯材、单板、胶合板、木质复合板、包装箱、集装箱、火柴杆和筷子等。最近10年杨木定向刨花板和结构复合板产量显著增加, 利用前景广阔。从资源生产的角度来看, 在改良木材化学组成、纤维质量和自然耐久性等方面进行木材性质改良的转基因杨树研究取得了长足进展。从资源利用角度来看, 高价值的工程复合板和高得率的纸浆和纸产品将会在未来发展中快速增长。