Functional analysis of a Douglas-fir metallothionein-like gene promoter: transient assays in zygotic and somatic embryos and stable transformation in transgenic tobacco

Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) metallothionein (PmMT) cDNA encodes a novel cysteine- and serine-rich MT, indicating a new subtype or prototype MT from which other plant MTs may have evolved. A genomic library of Douglas-fir was screened using MT cDNA probes, and genomic sequences that mediate tissue-specific, temporal as well as inducible expression of the embryo-specific MT-gene were analyzed. The promoter region of the PmMT genomic clone (gPmMT) contained a hexameric G-box, two putative ethylene-responsive elements and an inverted repeat of a motif similar to the core metal regulatory element. Interestingly, comparison of the upstream region of Douglas-fir gPm2S1 and gPmMTa genes revealed a conserved motif, CATTATTGA, not found in any known angiosperm gene promoter. Chimeric gene constructs containing a series of deletions in the gPmMTa promoter fused to the uidA reporter gene were assayed in Douglas-fir and transgenic tobacco (Nicotiana tabacum L.). Transient-expression assays in Douglas-fir megagametophyte and zygotic embryos indicated that the sequence –190 to +88 of gPmMTa was sufficient to drive the expression of the reporter gene and that the 225-bp fragment (–677 to –453) contained sequences necessary for high-level expression. In transgenic tobacco seedlings the -glucuronidase activity was localized in the vacuolar tissue and proliferating tissue of the auxiliary buds and stem elongation zone. The gPmMTa promoter was not active in the seeds of transgenic tobacco or in the roots of seedlings up to 3 weeks old. Detailed studies of transient expression and stable transformation provided important information on evolutionary conservation as well as novel features found in the conifer promoter. This is the first report of an MT-like gene promoter from conifers.     

Embryogenesis in flowering plants: Recent approaches and prospects

The overall architectural pattern of the mature plant is established during embryogenesis. Very little is known about the molecular processes that underlie embryo morphogenesis. Last decade has, nevertheless, seen a burst of information on the subject. The synchronous somatic embryogenesis system of carrot is largely being used as the experimental system. Information on the molecular regulation of embryogenesis obtained with carrot somatic embryos as well as observations on sandalwood embryogenic system developed in our laboratory are summarized in this review. The basic experimental strategy of molecular analysis mostly relied on a comparison between genes and proteins being expressed in embryogenic and non-embryogenic cells as well as in the different stages of embryogenesis. Events such as expression of totipotency of cells and establishment of polarity which are so critical for embryo development have been characterized using the strategy. Several genes have been identified and cloned from the carrot system. These include sequences that encode certain extracellular proteins (EPs) that influence cell proliferation and embryogenesis in specific ways and sequences of the abscisic acid (ABA) inducible late embryogenesis abundant (LEA) proteins which are most abundant and differentially expressed mRNAs in somatic embryos. That LEAs are expressed in the somatic embryos of a tree flora also is evidenced from studies on sandalwood. Several undescribed or novel sequences that are enhanced in embryos were identified. A sequence of this nature exists in sandalwood embryos was demonstrated using aCuscuta haustorial (organ-specific) cDNA probe. Somatic embryogenesis systems have been used to assess the expression of genes isolated from non-embryogenic tissues. Particular attention has been focused on both cell cycle and histone genes     

2S storage protein gene of Douglas-fir: characterization and activity of promoter in transgenic tobacco seeds.

To date a few sequences regulating expression of conifer seed-specific genes have been reported. To characterize Douglas-fir (Pseudotsuga menziesii [Mirb] Franco) 2S albumin storage protein genes, a genomic DNA sequence containing upstream promoter sequences was isolated by screening a Douglas-fir genomic library. Sequence analysis of the Douglas-fir gPm2S1 promoter revealed the presence of RY-repeated elements (GCATGC), and multiple E-box motifs (CANNTG) and ACGT-core elements, features characteristic of 2S storage protein genes in angiosperms. When fused to the GUS reporter gene, the 1.16 kb Douglas-fir 2S promoter sequence was sufficient to direct transient expression in both developing Douglas-fir embryos and maternally derived haploid megagametophytes. Analysis of this promoter construct in transgenic tobacco showed that expression was restricted to embryo and endosperm in developing seeds and was not detected in vegetative tissues of two-week-old seedlings. These results strongly suggest that both structural and regulatory elements as well as upstream signaling components controlling the expression of 2S albumin genes are highly conserved during evolution.     

Regulation of organogenesis and somatic embryogenesis by auxin in melon,Cucumis melo L.

Various tissues of seeds and seedlings of melon were cultured in vitro to study the effects of auxin concentration on organogenesis and embryogenesis. Adventitious shoots and somatic embryos were formed from explants of cotyledons of mature seeds, hypocotyls of seedlings, and leaves and petioles of young plantlets. Expanded cotyledons of seedlings formed only adventitious shoots. All tissues responded similarly to the 2,4-D concentration in the media, that is, adventitious shoots were formed at low concentration, callus proliferated without differentiation at intermediate concentration and somatic embryos were induced at high concentration. Cotyledons of mature seeds formed both adventitious shoots and somatic embryos more efficiently than any other tissues cultured.Effects of three auxins, 2,4-D, NAA and IAA, on organogenesis and embryogenesis were compared using cotyledons of mature seeds. Adventitious shoots were formed at low level of auxins (0 to 0.01 mg/l 2,4-D; 0 to 0.1 mg/l NAA; 0 to 1.0 mg/l IAA), and embryos were formed at high level of auxins (1.0 to 2.0 mg/l 2,4-D; 3.0 to 10.0 mg/l NAA; 20.0 to 100.0 mg/l IAA). IAA gave more efficient shoot formation and embryogenesis than the other auxins.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA 3indoleacetic acid - BA 6-benzylaminopurine - MS Murashige and Skoog     

银鲫原肠胚差异表达基因的筛选

胚胎发育是基因组中各个基因在时间和空间上选择性表达的结果。为了鉴定参与鱼类早期胚胎发育和胚层分化的调控因了,分别构建了银鲫(Carassiusauratusgibelio)原肠胚和成熟卵子的SMARTcDNA文库,并采用差异筛选的方法,从银鲫原肠胚SMARTcDNA文库中筛选不同于成熟卵子的差异表达基因。通过菌斑和PCR产物的两轮斑点杂交,从大约1500个克隆中筛选出131个阳性克隆,从中选择58个克隆测序并将测得的序列进行了数据库比对分析,结果显示这些差异表达基因大部分为参与转录和翻译的调控因子和核糖体蛋白以及一些在胚胎早期大量表达的参与脂类代谢的脂蛋白和一些未知的新基因。接着采用RTPCR技术,对其中6个基因在胚胎发育不同阶段的表达特征进行了分析,进一步证实这些基因在胚胎发育过程中存在表达差异;研究还从分析的基因中揭示出4类不同的表达模式。通过本研究,已筛选出一批在银鲫胚胎发育早期开始表达的调控基因,为开展鱼类胚胎发育早期表达基因的功能研究奠定了前期工作基础。       

Polyembryony in Citrus. Accumulation of seed storage proteins in seeds and in embryos cultured in vitro.

Citrus exhibits polyembryonic seed development, an apomictic process in which many maternally derived embryos arise from the nucellus surrounding the developing zygotic embryo. Citrus seed storage proteins were used as markers to compare embryogenesis in developing seeds and somatic embryogenesis in vitro. The salt-soluble, globulin protein fraction (designated citrin) was purified from Citrus sinensis cv Valencia seeds. Citrins separated into two subunits averaging 22 and 33 kD under denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cDNA clone was isolated representing a citrin gene expressed in seeds when the majority of embryos were at the early globular stage of embryo development. The predicted protein sequence was most related to the globulin seed storage proteins of pumpkin and cotton. Accumulation of 33-kD polypeptides was first detected in polyembryonic Valencia seeds when the majority of embryos were at the globular stage of development. Somatic Citrus embryos cultured in vivo were observed to initiate 33-kD polypeptide accumulation later in embryo development but accumulated these peptides at only 10 to 20% of the level observed in polyembryonic seeds. Therefore, factors within the seed environment must influence the higher quantitative levels of citrin accumulation in nucellar embryos developing in vivo, even though nucellar embryos, like somatic embryos, are not derived from fertilization events.     

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。     

Detection and seasonal expression pattern of a pathogenesis-related protein (PR-10) in Douglas-fir (Pseudotsuga menziesii) tissues

Previously, a PR-10 protein Pin m III was described in western white pine. In this study, primers based on cDNA of Pin m III were utilized to obtain the genomic sequence of a Pin m III homologue – Pse m I – in Douglas-fir. A comparative analysis of a deduced amino acid sequence of Pin m III and Pse m I genes indicated about 80% similarity between the two protein sequences, and a consensus 20 amino acid sequence located around the p-loop sequence was used to synthesize a peptide of 20 amino acids. An antibody to this synthetic peptide was able to detect the Pse m I protein in Douglas-fir. The anti- Pse m I antibody was used in a western immunoblot to monitor seasonal variation of the Pse m I in Douglas-fir needles and its level was shown to increase with overwintering of Douglas-fir seedlings. However, unlike the Pin m III, there is no indication that the Pse m I is associated with frost hardiness. Analysis of infected Douglas-fir roots showed a possible trend to up-regulation of Pse m I by pathogens such as the laminated root rot fungus, Phellinus weirii . The expression of Pse m I protein in Douglas-fir seedlings is very low compared to the expression of Pin m III protein in western white pine seedlings. In addition, a light-harvesting complex I protein, PSI-F, was identified in Douglas-fir by N-terminal amino acid sequencing.