落叶松体细胞胚成熟阶段差异表达的基因及部分基因的表达谱分析

摘要: 为研究落叶松体细胞胚胎发生的分子机理, 文章以日本落叶松×华北落叶松杂种无性系胚性细胞系Y35体细胞胚成熟阶段培养物的cDNA为实验组, 继代培养阶段胚性愈伤组织的cDNA为对照组, 利用抑制性消减杂交技术(Suppression subtractive hybridization, SSH)构建了体细胞胚成熟阶段的差异表达基因文库。随机选取800个阳性克隆进行测序, 共获得468个UniGenes, 共将其分为19类, 功能分析结果表明: 这些UniGenes可能参与代谢、转录、信号转导、转运、细胞生长分裂、细胞结构、细胞命运、蛋白质合成与降解、防御等与个体发育密切相关的生物学过程。对部分ESTs的表达谱进行分析, 结果表明这些ESTs均在落叶松体细胞胚胎发生的不同阶段特异表达。     

小叶杨Δ~1-吡咯琳-5-羧酸合成酶(P5CS)基因克隆及在杂种落叶松中的转化

以小叶杨为材料构建了干旱胁迫和正常生长条件下的cDNA文库,以特异性引物从中扩增出一条1 850bp大小的DNA片段,经序列分析证实该片段编码Δ1-吡咯琳-5-羧酸合成酶(P5CS)。将该片段构建入植物表达载体pBI121中,在落叶松杂交育种中利用花粉管通道法将带有该P5CS基因的植物表达质粒转化入杂种落叶松,收获球果取出种子,提取转化种子发芽长出幼芽的DNA,特异性PCR扩增和Southern,Western Blotting检测证实落叶松中已导入P5CS基因。     

Analysis of expression profile of selected genes expressed during auxin-induced somatic embryogenesis in leaf base system of wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and their possible interactions

Somatic embryogenesis is a notable illustration of plant totipotency and involves reprogramming of development in somatic cells toward the embryogenic pathway. Auxins are key components as their exogenous application recuperates the embryogenic potential of the mitotically quiescent somatic cells. In order to unravel the molecular basis of somatic embryogenesis, cDNA library was made from the regeneration proficient wheat leaf base segments treated with auxin. In total, 1440 clones were sequenced and among these 1,196 good quality sequences were assembled into 270 contigs and 425 were singletons. By reverse northern analysis, a total of 57 clones were found to be upregulated during somatic embryogenesis, 64 during 2,4-D treatment, and 170 were common to 2,4-D treatment and somatic embryogenesis. A substantial number of genes involved in hormone response, signal transduction cascades, defense, anti-oxidation, programmed cell death/senescence and cell division were identified and characterized partially. Analysis of data of select genes suggests that the induction phase of somatic embryogenesis is accompanied by the expression of genes that may also be involved in zygotic embryogenesis. The developmental reprogramming process may in fact involve multiple cellular pathways and unfolding of as yet unknown molecular events. Thus, an interaction network draft using bioinformatics and system biology strategy was constructed. The outcome of a systematic and comprehensive analysis of somatic embryogenesis associated interactome in a monocot leaf base system is presented. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The analysis of differential gene expression in early somatic embryogenesis on Lycium barbarum

Direct exposure of calluses of Lycium barbarum L. to an auxin-free medium can induce somatic embryogenesis. Somatic embryogenesis of Lycium barbarum L. is controlled artificially by regulating 2,4-D concentration. The total RNA that was isolated from calluses, embryonic calluses and early somatic embryos was used for analyzing differential genes expression. We obtained three cDNAs from early somatic embryogenesis which were not found in calluses. The results indicate that these cDNAs were early embryogenesis-specific cDNAs and this gene expression was induced in cultured calluses after a transfer to an auxin- free medium. A cDNA library was constructed using poly(A)⁺-RNA derived from early somatic embryos of Lycium barbarism L. Two full-length cDNAs were isolated from the library by differential screening. Northern blot hybridization analysis indicated that the expression of the full-length cDNA only existed in embryogenic calluses and early somatic embryos of Lycium barbarum L. This revised version was published online in June 2006 with corrections to the Cover Date.     

PyroClean: Denoising Pyrosequences from Protein-Coding Amplicons for the Recovery of Interspecific and Intraspecific Genetic Variation

High-throughput parallel sequencing is a powerful tool for the quantification of microbial diversity through the amplification of nuclear ribosomal gene regions. Recent work has extended this approach to the quantification of diversity within otherwise difficult-to-study metazoan groups. However, nuclear ribosomal genes present both analytical challenges and practical limitations that are a consequence of the mutational properties of nuclear ribosomal genes. Here we exploit useful properties of protein-coding genes for cross-species amplification and denoising of 454 flowgrams. We first use experimental mixtures of species from the class Collembola to amplify and pyrosequence the 5′ region of the COI barcode, and we implement a new algorithm called PyroClean for the denoising of Roche GS FLX pyrosequences. Using parameter values from the analysis of experimental mixtures, we then analyse two communities sampled from field sites on the island of Tenerife. Cross-species amplification success of target mitochondrial sequences in experimental species mixtures is high; however, there is little relationship between template DNA concentrations and pyrosequencing read abundance. Homopolymer error correction and filtering against a consensus reference sequence reduced the volume of unique sequences to approximately 5% of the original unique raw reads. Filtering of remaining non-target sequences attributed to PCR error, sequencing error, or numts further reduced unique sequence volume to 0.8% of the original raw reads. PyroClean reduces or eliminates the need for an additional, time-consuming step to cluster reads into Operational Taxonomic Units, which facilitates the detection of intraspecific DNA sequence variation. PyroCleaned sequence data from field sites in Tenerife demonstrate the utility of our approach for quantifying evolutionary diversity and its spatial structure. Comparison of our sequence data to public databases reveals that we are able to successfully recover both interspecific and intraspecific sequence diversity.     

Tandem repeat DNA localizing on the proximal DAPI bands of chromosomes in Larix, Pinaceae.

Repetitive DNA was cloned from HindIII-digested genomic DNA of Larix leptolepis. The repetitive DNA was about 170 bp long, had an AT content of 67%, and was organized tandemly in the genome. Using fluorescence in situ hybridization and subsequent DAPI banding, the repetitive DNA was localized in DAPI bands at the proximal region of one arm of chromosomes in L. leptolepis and Larix chinensis. Southern blot hybridization to genomic DNA of seven species and five varieties probed with cloned repetitive DNA showed that the repetitive DNA family was present in a tandem organization in genomes of all Larix taxa examined. In addition to the 170-bp sequence, a 220-bp sequence belonging to the same DNA family was also present in 10 taxa. The 220-bp repeat unit was a partial duplication of the 170-bp repeat unit. The 220-bp repeat unit was more abundant in L. chinensis and Larix potaninii var. macrocarpa than in other taxa. The repetitive DNA composed 2.0-3.4% of the genome in most taxa and 0.3 and 0.5% of the genome in L. chinensis and L. potaninii var. macrocarpa, respectively. The unique distribution of the 220-bp repeat unit in Larix indicates the close relationship of these two species. In the family Pinaceae, the LPD (Larix proximal DAPI band specific repeat sequence family) family sequence is widely distributed, but their amount is very small except in the genus Larix. The abundant LPD family in Larix will occur after its speciation.     

鸡枞菌转录组分析揭示其对木质纤维素的降解功能

【目的】探究鸡枞菌是否能降解木质纤维素成分,并理解其与共生白蚁之间的共生关系。【方法】本研究是应用新一代高通量测序技术454 GS FLX Titanium对鸡枞菌的转录组进行测序,挖掘鸡枞菌中能参与降解纤维素和木质素等成分的多样性酶系。【结果】八分之一的RUN测序总共得到了82386条表达序列标签,去除引物和载体等序列后,剩余的54410条序列被拼接成3301条contigs以及3193条singletons。根据序列相似性,将这些unigenes与三大蛋白数据库(Nr数据库、SwissProt数据库、CDD数据库)中的蛋白序列进行BLAST比较,发现有2681条基因与其他生物的已知基因有不同程度的相似性。在鸡枞菌的这些转录产物中,有33条编码可能参与降解纤维素或半纤维素的酶基因,其中包括5种纤维素酶以及28种水解半纤维素、淀粉或几丁质等物质的酶类。更重要的是,还发现了4种漆酶以及一种芳基乙醇氧化酶基因,这些都是能有效降解木质素的酶类。这些结果揭示了鸡枞菌中存在漆酶并可能有效降解植物残渣中的酚化合物。【结论】这些基因的发现说明了鸡枞菌能降解木质素,并能与共生白蚁分泌的纤维素酶协同作用有效降解纤维素。