Analysis of the rolC promoter region involved in somatic embryogenesis-related activation in carrot cell cultures.

In cell cultures of carrot (Daucus carota L.), somatic embryogenesis can be induced by transferring cells from a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) to one devoid of 2,4-D. Previous analysis of transgenic carrot cells containing the 5' non-coding sequence of the Ri plasmid rolC and a structural gene for bacterial beta-glucuronidase (uidA) has shown that the chimeric gene is actively expressed after induction of somatic embryogenesis. In this study, we demonstrate that activation of the rolC promoter is dependent on the process of embryo development but not on the duration of the cell culture in 2,4-D-free medium. We also analyzed the cis region of the rolC promoter that is responsible for somatic embryogenesis-related activation (SERA), namely relatively low beta-glucuronidase (GUS) activity in calli and proembryogenic masses (PEM) and high GUS activity in heart- and torpedo-stage embryos. When the -255-bp region of the rolC gene was used, SERA was retained. Internal deletions within this -255-bp region did not alter SERA by the rolC promoter. Furthermore, when a rolC promoter fragment (-848 to -94 bp) was fused to the cauliflower mosaic virus (CaMV) 35S core region (-90 to +6 bp), it conferred relatively low GUS activity in calli and PEM but high GUS activity in heart and torpedo embryos. When -848 to -255-bp or -255- to -94-bp fragments of the rolC promoter were fused to the same CaMV 35S core region, GUS activity patterns were not related to somatic embryogenesis. These results suggest that the combination of several regulatory regions in the rolC promoter may be required for SERA in carrot cell cultures.     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。     

从水稻细胞质型果糖-1,6-二磷酸酶启动子上游中去除93 bp可以彻底改变其表达模式

细胞质型果糖-1,6-二磷酸基因ATG上游1 195bp侧翼序列可调控GUS基因在水稻(Oryza sativa L.)中特异性表达,因此该片段包含有使报告基因在叶肉细胞中特异性表达的所有顺式元件.为了研究其调控特异表达的顺式元件,对启动子5′端进行了一系列的缺失,得到4种与GUS基因融合的植物表达载体,通过基因枪法转入水稻.结果表明,自启动子5′端-1 195 bp缺失至-1 102 bp时,GUS基因由叶肉细胞特异性表达变为组成型表达,且表达活性有所提高,推测在该区段中存在调控叶肉细胞特异性表达的顺式元件.进一步缺失仍然保持组成性表达的模式,即在转化株的根、茎和叶中的所有细胞中均有表达,同时启动子活性有所提高.这一结果暗示该启动子具有很大的应用潜力.     

水稻OsWTF1基因启动子的克隆与表达分析

目的：分析水稻OsWTF1基因启动子的功能及核心序列。方法：利用PCR技术从水稻日本晴基因组中克隆了转录因子WTF1编码区5上游大小为2049bp的调控区域,命名为OsWTF1,将它和长度为1631、608、474、415bp的5端缺失体分别与GUS基因融合构建表达载体,并用农杆菌介导法转化水稻。结果：GUS组织化学分析表明,OsWTF1、Os1631能够驱动GUS基因在根、茎、叶、叶鞘、花药、颖壳上的表达,Os608,Os474,Os415能驱动GUS在根、茎、花药、颖壳中表达,在叶鞘中未表达,而且在叶中的表达也很微弱。结论：OsWTF1启动子核心序列可能位于-1bp--415bp之间,在-608bp--1631bp之间可能存在与基因叶肉特异表达相关的重要元件。     

Investigation of the endosperm-specific sucrose synthase promoter from rice using transient expression of reporter genes in guar seed tissue

We report the investigation of an endosperm-specific promoter from the rsus3 gene from rice (Oryza sativa). The promoter was characterized by deletion analysis and transient expression in guar (Cyamopsis tetragonoloba) seed-tissue. Transient expression was monitored by histochemical GUS assay, and quantitative dual reporter assays comprising firefly luciferase as a test reporter, and Renilla luciferase and GUS as reference reporters. These revealed high expression levels of the reporter genes directed by the rsus3 promoter in guar endosperm. Specificity for this tissue in seeds was apparent from a virtual absence of reporter activity in guar cotyledons. Removal of a putative intron region only slightly raised the expression level, whereas duplication of the minimal promoter region, in a tandem-repeat rsus3 promoter construct, retained endosperm specificity in guar, and displayed three times the reporter activity observed with the single copy construct.     

Functional characterization of a putative nitrate transporter gene promoter from rice

Drought is one of the most significant abiotic stresses that influence plant growth anddevelopment.Expression analysis revealed that OsNRT1.3,a putative nitrate transporter gene in rice,wasinduced by drought.To confirm if the OsNRT1.3 promoter can respond to drought stress,a 2019 bpupstream sequence of OsNRT1.3 was cloned.Three OsNRT1.3 promoter fragments were generated by5′-deletion,and fused to the β-glucuronidase (GUS) gene.The chimeric genes were introduced into riceplants.NRT2019::GUS,NRT1196::GUS and NRT719::GUS showed similar expression patterns in seeds,roots,leaves and flowers in all transgenic rice,and GUS activity conferred by different OsNRT1.3 promoterfragments was significantly upregulated by drought stress,indicating that OsNRT1.3 promoter responds todrought stress and the 719 bp upstream sequence of OsNRT1.3 contains the drought response elements.     

Cloning of a new LEA1 gene promoter from soybean and functional analysis in transgenic tobacco

LEA1 gene from Glycine max can be expressed in late-embryo stage of plants, and respond to salinity and dehydration stress. To elucidate the mechanism for stress tolerance and high expression in seeds, we isolated and characterized the promoter of LEA1 gene (EQ, 1997 bp) starting the 5′LEA1 coding region. A deletion mutant of EQ promoter (ED) and the full length promoter (EQ) were fused to GUS reporter gene and transformed into the tobacco leaf discs. The results indicated that expression of the reporter gene (GUS) could be regulated by EQ promoter, and was stronger than the mutant under the stress conditions. Also, the expression level of GUS gene driven by EQ promoter in transgenic tobacco seeds was significantly higher than that by the mutant promoter, which meant that it had a better tissue-specificity. Therefore, the active domain for the promoter was located between ?1997 and ?1000 bp. Additionally, the activity of EQ promoter was 2.1-, 3.3- and 0.4- times stronger than the activity of promoter CaMV35S under salt (24 h), drought (10 h) or ABA (24 h), respectively. Meanwhile, the GUS activity of EQ promoter in seeds was 1.8-fold stronger compared to the promoter CaMV35S. In summary, the new promoter (EQ) is bi-functional, stress-inducible and seed-specific. These findings provide a further understanding for the regulation of LEA1gene expression, and suggest a new way for improving seed quality under saline and alkaline land.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Evidence for a role of β-1,3-glucanase in dicot seed germination

Class I β-1,3-glucanases are antifungal vacuolar proteins implicated in plant defense that show developmental, hormonal, and pathogenesis-related regulation. The expression was studied in germinating tobacco seeds of a chimeric β-glucuronidase (GUS) reporter gene fused to 1.6 kb of the 5' flanking sequence of the tobacco class I β-1,3-glucanase B (GLB) promoter. Histological staining for GUS activity showed that expression of the GLB promoter is highly localized in a specific zone of the endosperm in germinating seeds. The temporal and spatial patterns of GUS and β-1,3-glucanase activity found, suggest a novel function for class I β-1,3-glucanases during seed germination in a dicotyledonous plant.     

Visualization of somatic deletions mediated by R/<Emphasis Type="Italic">RS</Emphasis> site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.     

Functional dissection of a napin gene promoter: identification of promoter elements required for embryo and endosperm-specific transcription

The promoter region (?309 to +44) of the Brassica napus storage protein gene napA was studied in transgenic tobacco by successive 5′ as well as internal deletions fused to the reporter gene GUS (β-glucuronidase). The expression in the two main tissues of the seed, the endosperm and the embryo, was shown to be differentially regulated. This tissue-specific regulation within the seed was found to affect the developmental expression during seed development. The region between ?309 to ?152, which has a large effect on quantitative expression, was shown to harbour four elements regulating embryo and one regulating endosperm expression. This region also displayed enhancer activity. Deletion of eight bp from position ?152 to position ?144 totally abolished the activity of the napA promoter. This deletion disrupted a cis element with similarity to an ABA-responsive element (ABRE) overlapping with an E-box, demonstrating its crucial importance for quantitative expression. An internal deletion of the region ?133 to ?120, resulted in increased activity in both leaves and endosperm and a decreased activity in the embryo. Within this region, a cis element similar to the (CA)_n element, found in other storage protein promoters, was identified. This suggest that the (CA)_n element is important for conferring seed specificity by serving both as an activator and a repressor element.