Isolation and Characterization of Expressed Sequence Tags (ESTs) from Cambium Tissue of Birch (<Emphasis Type="Italic">Betula platyphylla Suk</Emphasis>)

Cloning and identification of genes from the cambium of woody plants are important steps in the investigation of wood formation. In order to clone and identify the genes involved in wood formation, a cDNA library was constructed from the cambial tissue of Betula platyphylla Suk (birch). From the cDNA library, 2,878 high-quality ESTs were generated, representing 1,540 tentative unique genes (TUGs). Approximately 70% of the ESTs were matched to proteins in the NR database in GenBank and were grouped into 12 functional categories according to their functional annotation. Fifty TUGs potentially involved in wood formation, including the process of lignin biosynthesis, cell wall structure, cell wall polysaccharides synthesis or development regulation, were identified from the EST collection. The time course expression analysis of 13 wood formation related genes was further studied using RNA gel blot and semi-quantitative RT-PCR. The results showed that these genes were primarily expressed in tissue that was collected during two time periods, spring (26 April to 11 June) and later summer (30 July to 18 Sept). The bimodal expression curve of the genes indicates that both of these time periods are critical in wood formation in birch, and may be associated with cell wall expansion earlier in the year, and cell wall strengthening later in the year.     

杉木木材形成过程特异表达基因的鉴定与分析

为了获得杉木木质化过程中特异表达的基因, 本研究利用抑制差减杂交技术, 以杉木突变体独干杉无性系为测试方(Tester), 正常的句容0号无性系为驱动方(Driver), 构建了正向差减文库, 获得了618个克隆。利用通用引物T7和SP6进行PCR及EcoRⅠ酶切鉴定文库重组子, 同时, 利用点杂交技术, 以正向差减、反向差减及未差减的测试方和驱动方四种探针, 进行了进一步的筛选阳性克隆。用定量PCR对结果进行了初步验证。260个单一ESTs中60%的与已知蛋白具有显著同源性, 可分为4个主要类别: 新陈代谢、细胞壁生成和结构重构、信号传导和胁迫。系统分析参与杉木木材形成的基因对了解木质部分化的分子机制具有重要意义, 是了解木材形成遗传控制的直接信息来源, 也是改良材形和纤维性质的潜在候选基因。     

Census of orthologous genes and self-organizing maps of biologically relevant transcriptional patterns in chickens (Gallus gallus)

The launch of large-scale chicken expressed sequence tags (EST) projects has placed the chicken in the lead for the number of EST sequences in agriculturally important animals. More than 451,000 chicken ESTs derived from over 158 libraries have been deposited in the NCBI dbEST database as of December 2003. But how many genes these ESTs represent and how they are expressed in different chicken tissues/organs remain undetermined. In the present research, we developed a human gene-based strategy for census of chicken orthologous genes and identification of their expression patterns. Among 34,157 human coding genes used in the study, BLAST analysis revealed that 11,066 genes provisionally matched 248,628 chicken ESTs. Based on the average EST abundance of the orthologous genes, the current public repository of chicken ESTs could represent 20,000 provisional genes. Analysis of gene expression in 14 single tissues/organs showed that approximately 15% of genes were expressed exclusively in single tissue/organ whereas the remaining 85% of genes were co-expressed in two or more tissues/organs. A majority (91.15%) of genes expressed in chicken embryos were also expressed at post-hatch stages, indicating that most genes activated in chicken embryos could serve housekeeping functions. Self-organizing maps (SOM) analysis organized 8807 provisional genes in selected chicken tissues into 98 clusters with each cluster being indicative of common regulatory factors and pathways. A total of 969 provisional orthologous genes were identified as preferentially expressed genes (PEGs) in various chicken tissues/organs (LOD>3.0). No doubt, the present study on gene expression patterns will provide insight into dynamics of metabolic pathways and tissue/organ programming and reprogramming in chickens.     

Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests.     

Expression profiling of the lignin biosynthetic pathway in Norway spruce using EST sequencing and real-time RT-PCR

Lignin biosynthesis is a major carbon sink in gymnosperms and woody angiosperms. Many of the enzymes involved are encoded for by several genes, some of which are also related to the biosynthesis of other phenylpropanoids. In this study, we aimed at the identification of those gene family members that are responsible for developmental lignification in Norway spruce (Picea abies (L.) Karst.). Gene expression across the whole lignin biosynthetic pathway was profiled using EST sequencing and quantitative real-time RT-PCR. Stress-induced lignification during bending stress and Heterobasidion annosum infection was also studied. Altogether 7,189 ESTs were sequenced from a lignin forming tissue culture and developing xylem of spruce, and clustered into 3,831 unigenes. Several paralogous genes were found for both monolignol biosynthetic and polymerisation-related enzymes. Real-time RT-PCR results highlighted the set of monolignol biosynthetic genes that are likely to be responsible for developmental lignification in Norway spruce. Potential genes for monolignol polymerisation were also identified. In compression wood, mostly the same monolignol biosynthetic gene set was expressed, but peroxidase expression differed from the vertically grown control. Pathogen infection in phloem resulted in a general up-regulation of the monolignol biosynthetic pathway, and in an induction of a few new gene family members. Based on the up-regulation under both pathogen attack and in compression wood, PaPAL2, PaPX2 and PaPX3 appeared to have a general stress-induced function. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.     

Regeneration of the secondary vascular system in poplar as a novel system to investigate gene expression by a proteomic approach

Wood formation is a complex process composing many biological events. To access its key developmental stages, we have established a regeneration system that can mimic the initiation and differentiation of cambium cells for Chinese white poplar. Anatomical studies showed that new cambium and xylem re-appeared in sequence within a few weeks after being debarked. This provides the opportunity to follow key stages of wood formation by sampling clonal trees at different regeneration times. We used this system in combination with a proteomic approach to analyze proteins expressed in different regeneration stages. PMFs for 244 proteins differentially displayed were obtained and queried against public databases. Putative functions of 199 of these proteins were assigned and classified. Regulatory genes for cell cycle progression, differentiation and cell fate were expressed in the formation of cambial tissue, while 27 genes involved in secondary wall formation were predominantly found in the xylem developing stage. This indicates that the change of gene expression pattern is corresponding to the progression of second vascular system regeneration when and where the key events of wood development occur. Further exploration of these interesting genes may provide insight into the molecular mechanisms of wood formation.