Differential,temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation

The temporal and spatial expression of oleosin and ₉-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height.In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins. Oil-body fractions prepared from young (2.5 mg) embryos contained very little oleosin protein, as examined by SDS-PAGE and western blotting, whereas identically prepared fractions from dry seeds contained over 10% (w/w) oleosin. Dehydration of oil bodies from young embryos resulted in their breakdown and coalescence into large clumps of oil which could not be re-emulsified, even after rehydration. In contrast, the oleosin-rich oil bodies from mature embryos were stable to dehydration and subsequent rehydration. It is suggested that, in developing rapeseed embryos, the accumulation of storage oil and oleosins is not concomitant but that the eventual deposition of oleosins onto the surfaces of storage oil bodies is essential for their stability during seed desiccation.Abbreviations ABA abscisic acid - ACP acyl carrier protein - GLC gas-liquid chromatography - PBS phosphate-buffered saline     

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4?×?44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.     

银鲫肌酸激酶M3-CK cDNA的克隆及其表达特征

用抑制性差减杂交结合SMART cDNA合成和RACE—PCR技术克隆到雌核发育银鲫(Carassius auratus gibelio)肌酸激酶M3-CK基因的全长cDNA。银鲫M3-CK cDNA全长1551bp，编码380个氨基酸，与普通鲤鱼(cyprinus carpio)M3-CK的氨基酸序列同源性高达95％。种系分析表明，银鲫M3-CK与其它脊椎动物的肌肉型肌酸激酶聚为较近的一支，与鲤鱼的M3-CK聚在一起，与脑特异型肌酸激酶及线粒体型肌酸激酶分歧较大。虚拟Northern杂交显示银鲫M3-CK基因在胚胎发育中差异表达。RT—PCR表明，银鲫M3-CK基因在成熟卵母细胞和胚胎发育早期可检测到少量的转录产物，在胚胎发育期间从肌肉效应期开始转录，并一直持续表达。组织RT—PCR表明，银鲫M3-CK基因只在心脏和肌肉表达。     

Identification and characterization of bone morphogenetic protein 2/4 gene from the starfish Archaster typicus

A bone morphogenetic protein 2/4 (BMP2/4) gene has been cloned from the starfish, Archaster typicus, for the purpose of investigating the expression pattern of the BMP4 gene in echinoderm embryos which do not produce micromeres. The isolated gene (named AtBMP2/4) contained two exons that encoded the entire coding region. The deduced AtBMP2/4 protein sequence contained 509 amino acids. Sequence comparison showed that it shared high amino acid similarity with sea urchin BMP2/4 and Xenopus BMP2 and BMP4. Northern blot analyses indicated that AtBMP2/4 mRNA initially appears at the blastula stage and has a maximal expression level at the gastrula stage. Whole-mount in situ hybridization revealed that AtBMP2/4 mRNA is expressed in the archenteron, coelomic vesicles, and ectodermal cells of gastrula stage embryos. The observed spatial distribution pattern vastly differs from that of sea urchin SpBMP2/4, which is expressed mainly in the oral ectoderm region of the mesenchyme blastula and early gastrula embryos.     

Isolation of the gene encoding Carrot leafy cotyledon1 and expression analysis during somatic and zygotic embryogenesis.

The Arabidopsis thaliana LEC1 gene regulates embryo morphology and seed maturation. For a better understanding of its function, we isolated a carrot (Daucus carota L. cv. US-Harumakigosun) counterpart of this gene, C-LEC1, from a cDNA library of carrot somatic embryos, since carrot is a better model plant for preparing large quantities of somatic embryos at the same developmental stage. The predicted amino acid sequence of C-LEC1 is similar to that of LEC1 and contains regions that are conserved in the heme-activated protein 3 (HAP3) subunit of plants, animals and microorganisms. C-LEC1 expression was detected in embryogenic cells, somatic embryos, and developing seeds. In situ hybridization analysis revealed C-LEC1 expression in the peripheral region of the embryos but not in the endosperm. Expression of C-LEC1 driven by Arabidopsis LEC1 promoter was able to complement the defects of the Arabidopsis lec1-1 mutant. These results suggest that C-LEC1 is a functional homolog of Arabidopsis LEC1, an important regulator of zygotic and somatic embryo development.     

新疆核桃WJ-CAD和ZJ-CAD基因克隆及时空表达表达分析

肉桂醇脱氢酶(CAD)是木质素生物合成中最后一步反应酶。本研究采用RT-PCR技术克隆露仁核桃WJ-CAD基因和硬壳完整核桃ZJ-CAD基因,研究CAD基因在硬壳完整和露仁核桃内果皮中发育过程中的表达特性。WJ-CAD基因cDNA序列含有788 bp ORF,编码258个氨基酸,分子量84.57 kD,理论等电点5.05;ZJ-CAD基因cDNA序列含有666 bp ORF,编码332个氨基酸,分子量81.93 kD,理论等电点5.09;对所编码的蛋白预测分析表明均属于FrmA超基因家族;实时荧光定量PCR结果表明,WJ-CAD基因的相对表达量整体呈现下降-上升-下降的趋势,ZJ-CAD基因的相对表达量整体均呈现上升-下降-上升-下降的趋势,2个基因均在花后65 d表达量达到最高峰,其后期表达量均较低,CAD基因在"温138"核桃内果皮中的表达量远低于同期"纸皮"的表达量。推测"温138"核桃露仁现象由于花后65 d后木质素合成量降低,导致缺乏木质素的积累或影响木质素单体的组成,从而造成内果皮发育不完整(露仁),初步证明CAD基因参与调控木质素合成,露仁现象可能由于硬核期缺乏木质素的积累。