The expression of a potato (Solanum tuberosum) S-RNase gene in Nicotiana tabacum pollen

Self-incompatibility in the Solanaceae is controlled by a single multiallelic genetic locus, the S locus. The stylar gene products of the S locus are abundant glycoproteins with ribonuclease activity, secreted in the transmitting tract tissue of the pistil. To investigate the structural and functional integrity and possible phenotypic effects of expression of the S-gene product in the male gametophyte, N. tabacum plants were transformed with a construct containing the genomic S ₂ -RNase coding sequence from S. tuberosum under the control of the promoter of the pollen-specific LAT52 gene from tomato. The expression pattern of the S ₂ RNase in the male gametophyte at both the protein and RNA level was found to be identical to that already reported for expression of the -glucuronidase (GUS) gene directed by the LAT52 promoter in transgenic tomato and tobacco. The S ₂ -RNase gene fusion led to a tissue-specific and developmentally regulated accumulation of the S ₂ polypeptide in pollen of transgenic tobacco plants. The transgenic protein product was of the same size and charge as the potato stylar product, had ribonuclease activity, and was glycosylated. The transgenic plants, however, did not show any morphological variations in their flower organs, and their fertility was not influenced by the accumulation of the S ₂ -RNase protein in pollen.     

Multiple elements of the S 2 ?-RNase promoter from potato ( Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position −200 to bp −100, and motif II, located between bp −498 and −480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues. Received: 7 August 1997 / Accepted: 8 September 1997     

Expression of three <Emphasis Type="Italic">Arabidopsis</Emphasis> cytokinin oxidase/dehydrogenase promoter::GUS chimeric constructs in tobacco: response to developmental and biotic factors

Expression patterns of three Arabidopsis thaliana cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions were investigated in tobacco plants. While cytokinin oxidase/dehydrogenase promoter 2 showed no expression in tobacco, the cytokinin oxidase/dehydrogenase promoters 3 and 4 were active in various tissues throughout development of the tobacco. Recently, the 1452 bp promoter region of AtCKX3 was reported as almost inactive in Arabidopsis. In contrast, the 1627 bp DNA fragment preceding the AtCKX3 coding region drove expression of the reporter GUS gene in various tobacco tissues. The promoter was mainly expressed in tobacco leaves and roots during early stages of development but also later in young flower buds as well as in pollen grains. The construct was particularly active before (hypocotyl region) and during (vascular system) lateral root initiation, supporting the idea of an inhibitory role of active cytokinins in the process of root initiation. The cytokinin oxidase/dehydrogenase promoter 4::GUS fusion in tobacco was shown to share some common (but weaker) expression patterns with promoter 3, namely in the leaves and pollen, but also conferred specific expression in tobacco root cap cells and trichomes. In addition, the response of cytokinin oxidase/dehydrogenase promoter::GUS reporter fusions to infection with the leafy gall-forming bacteria Rhodococcus fascians was examined. While an avirulent strain of R. fascians did not induce expression of any of the cytokinin oxidase/dehydrogenase promoters, the cytokinin oxidase/dehydrogenase promoter 3::GUS fusion was specifically induced at the site of infection when plants were challenged with a virulent strain of R. fascians, providing a possible explanation for the lack of significantly elevated cytokinin concentrations in tissues infected with virulent strains of R. fascians.This revised version was published online in August 2005 with some black and white figures replaced by coloured figures.     

Characterization of a Novel Pollen-Specific Promoter from Wheat (Triticum Aestivum L.)

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the ??-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4?kb fragment of the promoter was enough to confer pollen-specific expression.     

Developmental regulation of an acyl carrier protein gene promoter in vegetative and reproductive tissues

The expression of an Arabidopsis acyl carrier protein (ACP) gene promoter has been examined in transgenic tobacco plants by linking it to the reporter gene -glucuronidase (GUS). Fluorometric analysis showed that the ACP gene promoter was most active in developing seeds. Expression was also high in roots, but significantly lower in young leaves and downregulated upon their maturation. Etiolated and light-grown seedlings showed the same level of GUS activity, indicating that this promoter is not tightly regulated by light. Histochemical studies revealed that expression was usually highest in apical/ meristematic zones of vegetative tissues. Young flowers (ca. 1 cm in length) showed GUS staining in nearly all cell types, however, cell-specific patterns emerged in more mature flowers. The ACP gene promoter was active in the stigma and transmitting tissue of the style, as well as in the tapetum of the anther, developing pollen, and ovules. The results provide evidence that this ACP gene is regulated in a complex manner and is responsive to the array of signals which accompany cell differentiation, and a demand for fatty acids and lipids, during organogenesis.     

Features of the hmg 1 subfamily of genes encoding HMG-CoA reductase in potato

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) catalyzes a key step in isoprenoid metabolism leading to a range of compounds that are important for the growth, development and health of the plant. We have isolated 7 classes of genomic clones encoding HMGR from a potato genomic library. Comparison of nucleic acid sequences reveals a high degree of identity between all seven classes of clones and the potato hmg 1 gene described by Choi et al. (Plant Cell 4: 1333, 1992), indicating that all are members of the same subfamily in potato. A representative member (hmg 1.2) of the most abundant class of genomic clones was selected for further characterization. Transgenic tobacco and potato containing the -glucuronidase (GUS) reporter gene under the control of the hmg 1.2 promoter expressed GUS activity constitutively at a low level in many plant tissues. High levels of GUS activity were observed only in the pollen. GUS assays of isolated pollen, correlations of GUS activity with the HMGR activity of anthers, hmg 1.2 promoter deletion studies, and segregation analysis of the expression of hmg 1.2::GUS among the R₂ pollen of R₁ progeny plants demonstrated that the hmg 1.2 promoter controls pollen expression.     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

Gene expression and oxalate oxidase activity of two germin isoforms induced by stress

Wheat germin is a homopentameric 125 kD glycoprotein mainly localized in the cell wall of monocots, and is a specific marker of the onset of growth in germinating seeds. The major objective of this study was to examine the expression and oxalate oxidase activity of two wheat germin isoforms: gf-2.8 and gf-3.8 in transgenic tobacco plants. The transgenic tobacco plants were created with different constructs: 1) one entire excision of gf-2.8 germin promoter and two partially deleted promoter sequences were used to generate 3 independent GUS constructs; 2) the whole gf-2.8 gene construct and the fusion with CaMV 35S promoter; 3) one entire excision of gf-3.8 germin gene and one partially deleted gf-3.8 promoter sequences were used to generate 2 independent GUS constructs; 4) the whole gf-3.8 gene and the fusion with CaMV 35S promoter. Hormonal treatment (auxin and gibberellin), salt treatment, heavy metals (Mn, Fe, Co, Ni, Cu, Zn, Cd, Hg, As) and Al induced high GUS activity in tobacco transformed with entire and one partially deleted of the gf-2.8 gene. The immunoblotting confirmed induction of gf-2.8 gene and its product expressed oxalate oxidase activity in tobacco transformed with the entire gf-2.8 construct. Neither nicotinic acid, salicylic acid, heat shock, cold nor UV-C have enhanced significant GUS activity and germin gf-2.8 synhesis and activity. The germin gf-3.8 constructs with GUS gene and with the entire gf-3.8 sequences gave non-positive response with factors mentioned above. It has been demonstrated that gf-3.8 germin isoform is present as a monomer (M_r 25 kD). The non-active gf-3.8 protein is synthetised in transgenic tobacco plants only under control of the CaMV 35S promoter. Consequently, among two germin isoforms, only the gf-2.8 protein seems to be regulated by hormonal, salt and heavy metal factors. The gf-2.8 oxalate oxidase activity could be then involved in general stress-induced signalling in plant.     

An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum

In many flowering plants, self-fertilization is prevented by an intraspecific reproductive barrier known as self-incompatibility (SI), that, in most cases, is controlled by a single multiallelic S locus. So far, the only known S locus product in self-incompatible species from the Solanaceae, Scrophulariaceae and Rosaceae is a class of ribonucleases called S RNases. Molecular and transgenic analyses have shown that S RNases are responsible for pollen rejection by the pistil but have no role in pollen expression of SI, which appears to be mediated by a gene called the pollen self-incompatibility or Sp gene. To identify possible candidates for this gene, we investigated the genomic structure of the S locus in Antirrhinum, a member of the Scrophulariaceae. A novel F-box gene, AhSLF-S ₂, encoded by the S ₂ allele, with the expected features of the Sp gene was identified. AhSLF-S ₂ is located 9 kb downstream of S ₂ RNase gene and encodes a polypeptide of 376 amino acids with a conserved F-box domain in its amino-terminal part. Hypothetical genes homologous to AhSLF-S ₂ are apparent in the sequenced genomic DNA of Arabidopsis and rice. Together, they define a large gene family, named SLF (S locus F-box) family. AhSLF-S ₂ is highly polymorphic and is specifically expressed in tapetum, microspores and pollen grains in an allele-specific manner. The possibility that Sp encodes an F-box protein and the implications of this for the operation of self-incompatibility are discussed.     

Multiple elements of the S 2 -RNase promoter from potato (Solanum tuberosum L.) are required for cell type-specific expression in transgenic potato and tobacco

A functional analysis of the promoter of the S ₂ -RNase gene from potato was performed in transgenic potato and tobacco plants, using a deletion series of S ₂ -RNase promoter GUS fusions. A detailed histochemical and quantitative analysis of the transgenic tobacco plants revealed that S ₂ promoter fragments ranging in size from 5.6 kb in length down to 0.2 kb mediate a weak developmentally regulated expression in the pistil, and strong ectopic expression in pollen. In the pistil, different expression patterns were seen depending on the transformant, the predominant one being characterised by expression in the stigma and the transmitting tract of the style, whereas a few plants showed expression exclusively either in the stigma or in the stylar transmitting tissue. All transformants also showed GUS expression in the placental epidermis of the ovary. Two sequences that are conserved between the potato S ₁ -RNase and S ₂ -RNase promoters, termed motif I and motif III, are located in a fragment of the S ₂ promoter extending from position ?200 to bp ?100, and motif II, located between bp ?498 and ?480, was identified on the basis of sequence comparisons between pistil-specific promoters. Motif II was found to be dispensible for pistil-specific and for pollen-specific expression. Two submotifs, A and B, were identified within motif I. Both were essential for expression in the pistil but only B was necessary for expression in pollen. Although motif III has a similar bipartite structure and sequence to motif I, it was not sufficient to confer either pollen- or pistil-specific expression. However, deletion of motif III abolished pollen-specific expression in transient expression experiments, suggesting that an interaction between the two sequence motifs may be needed to specify cell type-specific expression. In transgenic potato the S ₂ -RNase promoter also mediates expression in pollen and in the pistil; however, significantly fewer plants showed expression than in tobacco, with most plants also exhibiting GUS expression in other tissues.     

竹节花黄斑驳病毒启动子的缺失分析及功能

竹节花黄斑驳病毒(CoYMV)是侵染单子叶植物竹节花的一 种双链环状DNA病毒,它的启动子可介导外源基因在烟草韧皮部特异表达。为了研究其组织 特异性表达的最佳启动子区域,对CoYMV启动子进行了5′端五种不同长度的缺失分析,用不同长度的启动子片段与GUS基因及NOS3′端转录中止序列构建了全长启动子及5 个缺失启动子序列的六个嵌合GUS基因植物表达载体。利用农杆菌将上述嵌合基因转化烟草 外植体后,每种表达载体都获得了一批转基因烟草植株。转化再生烟草植株的PCR分析、GUS 酶活测定及GUS组织染色的结果表明六种类型的嵌合基因已整合到烟草染色体中,并有五种 表达出GUS活性。缺失到870bp的启动子比全长启动子(1040bp)的活性约高78%,870bp比585bp启动子介导的GUS活性略高但差别不明显,缺失到447和232时GUS活性有明显下 降,但仍具有韧皮部特异表达的特性。当缺失到TATA box附近的44bp时启动子丧失组织特 异性,GUS活性也降低到测不出来的水平。以上结果表明CoYMV启动子从转录起始位点上游 870bp～230bp及232bp下游区分别与启动子的活性和韧皮部组织特异性密切相关,870bp上游可能存在一个负调控序列,所以该启动子的活性和组织特异性的最佳调控区应在87 0bp或585bp的下游区。CoYMV启动子与35S启动子驱动GUS基因在烟草中表达的活性相比, 前者为后者的70%左右,考虑到前者仅在韧皮部细胞表达而后者为组成型表达,所以CoYMV启 动子在韧皮部的活性可能与35S启动子相当或更高。CoYMV启动子在其它转基因植物中驱动外 源基因表达的特点正在研究中。     

Spacer length-dependent protection of specific activity of pollen and/or embryo promoters from influence of CaMV 35S promoter/enhancer in transgenic plants

The influence of the CaMV 35S promoter/enhancer on expression profiles of four Arabidopsis thaliana pollen- and/or embryo-specific promoters, APRS, ESL, MXL, and DLL, was tested in transgenic tobacco plants. Individual promoters were fused to the gus reporter gene and cloned in head-to-head orientation with the CaMV 35S:hpt expression unit within the same T-DNA. With the exception of the TATA-less promoter DLL, all other combinations generated interactions between the promoter under investigation and 35S promoter/enhancer resulting in ectopic β-glucuronidase (GUS) expression in vegetative organs and tissues, the most susceptible being the stem, followed by callus, leaf, and root. To eliminate this crosstalk, DNA spacers of length 1, 2 and 5 kb were cloned between the interacting sequences. Ectopic GUS staining was dependent on the affected promoter as well as the distance between the 5′-end of the CaMV 35S promoter and the reporter gene translation start site. When this distance was less than 1 kb strong ectopic GUS staining was observed in all vegetative tissues, similar to the CaMV35S:gus expression profile in transgenic tobacco plants. Insertion of spacer DNA sequences of increasing length resulted in gradual reduction of ectopic GUS staining in tested plants. Of the tissues and organs related to plant reproduction, only anthers and seed coats in the early stages of seed development showed ectopic GUS staining. Developing pollen and embryos showed a pattern of GUS activity consistent with the predicted role of a developmental stage-specific promoter in transgenic tobacco plants.     

Cotton α-Globulin Promoter: Isolation and Functional Characterization in Transgenic Cotton,Arabidopsis, and Tobacco

Globulins are the most abundant seed storage proteins in cotton and, therefore, their regulatory sequences could potentially provide a good source of seed-specific promoters. We isolated the putative promoter region of cotton -globulin B gene by gene walking using the primers designed from a cotton staged embryo cDNA clone. PCR amplified fragment of 1108 bp upstream sequences was fused to gusA gene in the binary vector pBI101.3 to create the test construct. This was used to study the expression pattern of the putative promoter region in transgenic cotton, Arabidopsis, and tobacco. Histochemical GUS analysis revealed that the promoter began to express during the torpedo stage of seed development in tobacco and Arabidopsis, and during cotyledon expansion stage in cotton. The activity quickly increased until embryo maturation in all three species. Fluorometric GUS analysis showed that the promoter expression started at 12 and 15 dpa in tobacco and cotton, respectively, and increased through seed maturation. The strength of the promoter expression, as reflected by average GUS activity in the seeds from primary transgenic plants, was vastly different amongst the three species tested. In Arabidopsis, the activity was 16.7% and in tobacco it was less than 1% of the levels detected in cotton seeds. In germinating seedlings of tobacco and Arabidopsis, GUS activity diminished until it was completely absent 10 days post imbibition. In addition, absence of detectable level of GUS expression in stem, leaf, root, pollen, and floral bud of transgenic cotton confirmed that the promoter is highly seed-specific. Analysis of GUS activity at individual seed level in cotton showed a gene dose effect reflecting their homozygous or hemizygous status. Our results show that this promoter is highly tissue-specific and it can be used to control transgene expression in dicot seeds.     

Transgene Expression in the Vegetative Tissues of Apple Driven by the Vascular-specific rolC and CoYMV Promoters

The ability of the heterologous promoters, rolCP and CoYMVP, to drive expression of the gusA reporter gene in the vegetative tissues of apple (Malus pumila Mill.) has been studied using transgenic plants produced by Agrobacterium-mediated transformation. Replicate plants of each transgenic clone were propagated in soil to a uniform size and samples of leaf, petiole, stem, and root were taken for the measurement of -glucuronidase (GUS) activity by fluorometric assay. The levels of expression were compared with those in tissues of a representative clone containing the CaMV 35S promoter. These quantitative GUS data were related to the copy number of transgene loci assessed by Southern blotting. The CoYMV promoter was slightly more active than the rolC promoter, although both expressed gusA at a lower level than the CaMV 35S promoter. In clones containing the rolC promoter with multiple transgene loci, expression values were generally among the highest or lowest in the range. The precise location of GUS activity in each tissue was identified by staining of whole leaves and tissue sections with a chromogenic substrate. This analysis demonstrated that with both the rolC and CoYMV promoters the reporter gene activity was primarily localised to vascular tissues, particularly the phloem. Our results indicate that both promoters would be suitable to drive the expression of transgenes to combat pests and diseases of apple that are dependent on interaction with the phloem.     

Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis

To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the beta-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate co-ordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.