A rice promoter containing both novel positive and negative cis-elements for regulation of green tissue-specific gene expression in transgenic plants

The tissue-specific expression of transgenes is essential in plant breeding programmes to avoid the fitness costs caused by constitutive expression of a target gene. However, knowledge on the molecular mechanisms of tissue-specific gene expression and practicable tissue-specific promoters is limited. In this study, we identified the cis -acting elements of a tissue-specific promoter from rice, P_D54O , and tested the application of original and modified P_D54O and its cis -elements in the regulation of gene expression. P_D54O is a green tissue-specific promoter. Five novel tissue-specific cis -elements (LPSE1, LPSE2, LPSRE1, LPSRE2, PSE1) were characterized from P_D54O . LPSE1 activated gene expression in leaf and young panicle. LPSRE2 suppressed gene expression in leaf, root, young panicle and stem, and PSE1 suppressed gene expression in young panicle and stem. LPSRE1 and LPSE2 had dual roles in the regulation of tissue-specific gene expression; both functioned as activators in leaf, but LPSRE1 acted as a repressor in stem and LPSE2 as a repressor in young panicle and root. Transgenic rice plants carrying cry1Ac encoding Bacillus thuringiensis endotoxin, regulated by P_D54O , were resistant to leaf-folders, with no Cry1Ac protein found in endosperm or embryo. A reporter gene regulated by a series of truncated P_D54O showed various tissue-specific expression patterns. Different fragments of P_D54O fused with the constitutive cauliflower mosaic virus 35S promoter suppressed 35S -regulated gene expression in various tissues. P_D54O , truncated P_D54O and the tissue-specific cis -elements provide useful tools for the regulation of tissue-specific gene expression in rice breeding programmes.     

Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat

Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi‐1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for β‐glucuronidase (GUS), under the control of the maize Ubi‐1 promoter in individual transgenic wheat (Triticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi‐1 promoter had strong activity in young, metabolically active tissues and in pollen grains.     

A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to ?1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (?1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5′ sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, ?908H3/GUS and ?185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from ?909 to ?1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to ?185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.     

A class II patatin promoter is under developmental control in both transgenic potato and tobacco plants

Summary A new member of the patatin gene family belonging to the class II subfamily was isolated and characterized by DNA sequencing. In order to study the expression profile of this gene, the promoter was fused to the -glucuronidase gene and transferred to potato and tobacco. Histochemical analysis revealed high expression in a few defined cells in potato tubers and in a specific layer of both potato and tobacco root tips. In contrast to the developmentally and metabolically regulated class I patatin gene B33 this gene was not inducible by elevated levels of sucrose. Expression of this chimaeric gene was also found in callus and suspension cultures of potato.     

Upstream sequences regulating legumin gene expression in heterologous transgenic plants

Summary We have previously isolated a legumin gene LeB4 from Vicia faba and shown that a 4.7 kb DNA fragment containing the gene leads to seed-specific expression in transgenic tobacco plants. Here we report that the 2.4 kb upstream sequence alone, when fused to either the neomycin phosphotransferase II (nptII) gene or the -glucuronidase (uidA) gene, leads to high enzyme levels in transgenic seeds of both tobacco and Arabidopsis. -Glucuronidase (GUS) activity is especially intense in the cotyledons fading out towards the embryonal root tip, a result confirmed by in situ hybridization. Staining of endosperm cells is consistent in both species. Analysis of a series of promoter deletion mutants fused to the nptII gene and introduced into tobacco plants revealed that about 1 kb of 5-flanking sequence is sufficient for high-level expression but indirect evidence suggests the presence of weak positive regulatory elements further upstream. Deletions leaving only 0.2 kb of upstream sequence reduce enzyme levels to less than 10%. A deletion which destroys the legumin box with its seed protein gene-specific CATGCATG motif has no obvious effects on expression levels.     

Anther-specific expression of ipt gene in transgenic tobacco and its effect on plant development

Isopentenyl transferase (ipt) gene from Agrobacterium tumefaciens T-DNA was placed under the control of a TA29 promoter which expresses specifically in anther. The chimeric TA29-ipt gene was transferred to tobacco plants. During flowering, mRNA of the ipt gene in the anthers of the transgenic plants accumulated and the level of iPA + iPs increased 3–4-fold in the leaves, petals, pistils, and stamens compared with those in the wild type plants. This cytokinin increase affected various aspects in development indicating that the alterations of endogenous cytokinin level by using anther-specific expression of the TA29-ipt gene affected morphology, floral organ systems and reproductivity of the transgenic plants.     

水稻谷蛋白启动子驱动下的ipt基因在转基因烟草中的表达

利用农杆菌系统介导 ,采用叶盘转化法 ,将在水稻谷蛋白启动子驱动下的外源ipt基因导入烟草植株中 ,经过抗生素筛选、PCR与测序分析检测出转基因植株。成熟的转基因烟草种子经过ELISA细胞分裂素试剂盒检测 ,发现iPAs含量为对照的 2 .43倍 ,此外 ,种子的重量也增加了 7.8%。     

Glufosinate-tolerant tobacco plants directed by the promoter of adenylate kinase gene of rice

A DNA clone containing the 5' part of the adenylate kinase (AK) gene was isolated from a rice genomic library, and its nucleotide sequence was determined. This clone consists of 5' upstream, five exons and four introns of the AK gene. All of the determined donor and receptor sites contained 'GT' and 'AG' consensus splice sequences. Transgenic tobacco plants harbouring a chimeric gene consisting of the 5' upstream sequence of the AK gene fused with the gene encoding phosphinothricin acetyl transferase were generated. They showed tolerance to glufosinate to a level four times higher than its commercial dose.     

Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants

Summary In order to study possible functions of the inclusion body matrix protein (IBMP) encoded by gene VI of cauliflower mosaic virus (CaMV), the XbaI fragment containing the gene VI of a Japanese strain of CaMV (CaMV S-Japan) was transferred to tobacco plants by Ti mediated transformation. Eight out of 18 kanamycin resistant plants (40%) expressed detectable levels of IBMP. Those transgenic plants expressing IBMP produced leaves with light green color, and their growth was suppressed as compared with control plants. Symptom-like necrotic spots also appeared on the leaves and stems of the mature transgenic plants. Furthermore, in these transgenic plants, pathogenesis-related proteins 1a, 1b and 1c were highly expressed and the activity of 1,3--glucanase was increased up to eightfold. From these results, we concluded that expression of the IBMP is associated with symptom development.     

Agrobacterium-mediated transformation of monocot and dicot plants using the NCR promoter derived from soybean chlorotic mottle virus

The NCR promoter (PNCR) from soybean chlorotic mottle virus (SoyCMV) was used to express the selectable marker, neomycin phosphotransferase (nptII) gene, in Agrobacterium-mediated transformation of both monocot (rice) and dicot (tobacco) plants. A multi-cloning site for insertion of a gene of interest into the binary vector pTN is located proximal to the right border region of T-DNA. When chimeric genes under the control of other strong promoters were located in a head-to-head orientation to the PNCR-nptII gene, kanamycin-resistant tobacco shoots were generated more efficiently than when using the original pTN vectors. This suggests that the enhancer-like sequences in the promoters adjacent to PNCR may promote expression of the PNCR-nptII gene. Received: 20 August 1999 / Revision received: 16 November 1999 / Accepted: 19 November 1999     

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.