Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther’s tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species. Data deposition: The sequence reported in this paper have been deposited in the GeneBank database (Accession No. U12171)     

Promoters of two anther-specific genes confer organ-specific gene expression in a stage-specific manner in transgenic systems

Differential screening of a stage-specific cDNA library of Indica rice has been used to identify two genes expressed in pre-pollination stage panicles, namely OSIPA and OSIPK coding for proteins similar to expansins/pollen allergens and calcium-dependent protein kinases (CDPK), respectively. Northern analysis and in situ hybridizations indicate that OSIPA expresses exclusively in pollen while OSIPK expresses in pollen as well as anther wall. Promoters of these two anther-specific genes show the presence of various cis-acting elements (GTGA and AGAAA) known to confer anther/pollen-specific gene expression. Organ/tissue-specific activity and strength of their regulatory regions have been determined in transgenic systems, i.e., tobacco and Arabidopsis. A unique temporal activity of these two promoters was observed during various developmental stages of anther/pollen. Promoter of OSIPA is active during the late stages of pollen development and remains active till the anthesis, whereas, OSIPK promoter is active to a low level in developing anther till the pollen matures. OSIPK promoter activity diminishes before anthesis. Both promoters show a potential to target expression of the gene of interest in developmental stage-specific manner and can help engineer pollen-specific traits like male-sterility in plants. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Accessions: OSIPA cDNA, AF220610; OSIPK cDNA, AF312920; OSIPA partial gene and upstream promoter region, AY166659; OSIPK gene-specific and upstream sequence, AY168440.     

The promoter-elements of some abiotic stress-inducible genes from cereals interact with a nuclear protein from tobacco

In this communication, we report the binding of abscisic acid responsive elements (ABREs) of rice Osem, namely motif A and motif B, with a cognate trans-acting factor present in the nuclear extract of tobacco leaf. The binding is specific as both the complexes were disrupted with an excess of homologous non-radioactive DNA like motif A or motif B themselves or with cis-elements of rice Rab16A, motif I (ABRE) and motif IIa (non-ACGT ABRE-like sequences). Four tandem repeats of ABRE from wheat Em (4X ABRE) or two tandem repeats of Em ABRE, plus two copies of coupling element (CE1) from barley HVA22 (2X ABRC), also showed specific complexes, that were competed out by an excess of homologous competitors like motif I, motif IIa, motif A, motif B, 4X ABRE and 2X ABRC, but not by the unrelated 4X DRE sequence. Elution of the protein from all the complexes showed a single 26 kDa polypeptide band. Introgression of two of the above synthetic promoters 4X ABRE and 2X ABRC, each fused with minimal promoter of cauliflower mosaic virus 35S (CaMV 35S), could induce the expression of the reporter gene β-glucuronidase (gus) in transgenic tobacco in response to high NaCl concentration, dehydration or abscisic acid, but not at the constitutive level, proving that they can be used as efficient stress-inducible promoters. Our work shows both in vivo and in vitro activity of the promoters from monocot genes in the model dicot plant tobacco.     

熊蜂生假丝酵母启动子的筛选及强度分析

【背景】熊蜂生假丝酵母(Starmerella bombicola)作为一种非常规假丝酵母菌株,因其具备高产槐糖脂生物表面活性剂的能力而受到广泛关注。然而,由于自身的表达系统并不完善,限制了该菌株的代谢工程改造。【目的】克隆、筛选及鉴定新的系列内源启动子表达元件。【方法】通过对比分析熊蜂生假丝酵母全基因组及9种功能已知目的基因信息,并结合启动子预测网站,筛选获得系列启动子候选序列,以SbGFP (密码子优化后的酵母增强型绿色荧光蛋白)为报告基因进行整合表达,通过绿色荧光蛋白强度及转录水平分析鉴定启动子强度。【结果】在分别以葡萄糖和油酸作为唯一碳源的条件下,启动子PTEF1和PGPD在不同碳源培养条件下均显示出较高的转录水平。启动子PCYP52M1、PUGTA1、PUGTB1及PMOB在以油酸为唯一碳源时具有弱转录活性,而在以葡萄糖为唯一碳源时则未检测到它们具有转录活性,推测它们是油酸诱导型启动子。进一步利用实时荧光定量PCR (RT-qPCR)对SbGFP进行转录水平分析,检测结果与绿色荧光表达水平一致。【结论】获得了系列熊蜂生假丝酵母内源性启动子,进一步丰富了该菌株的表达元件,为菌株的代谢工程改造及基因的表达与调控奠定了理论基础。     

A complex ensemble of cis-regulatory elements controls the expression of a Vicia faba non-storage seed protein gene

We have identified cis-regulatory elements within the 5-upstream region of a Vicia faba non-storage seed protein gene, called usp, by studying the expression of usp-promoter deletion fragments fused to reporter genes in transgenic tobacco seeds. 0.4 kb of usp upstream sequence contain at least six, but probably more, distinct cis-regulatory elements which are responsible for seemingly all quantitative, spatial and temporal aspects of expression. Expression-increasing and-decreasing elements are interspersed and include an AT-rich sequence, a G-box element and a CATGCATG motif. The latter acts as a negative element in contrast to what has been found for the same motif in legumin-and vicilin-type seed storage protein gene promoters. Seed specificity of expression is mainly determined by the –68/+51 region which confers, however, only very low levels of expression. The data support the combinatiorial model of promoter function.     

Promoters of nuclear‐encoded respiratory chain Complex I genes from Arabidopsis thaliana contain a region essential for anther/pollen‐specific expression

Regulatory promoter regions responsible for the enhanced expression in anthers and pollen are defined in detail for three nuclear encoded mitochondrial Complex I (nCI) genes from Arabidopsis thaliana. Specific regulatory elements were found conserved in the 5′ upstream regions between three different genes encoding the 22 kDa (PSST), 55 kDa NADH binding (55 kDa) and 28 kDa (TYKY) subunits, respectively. Northern blot analysis and transgenic Arabidopsis plants carrying progressive deletions of the promoters fused to the β-glucuronidase (GUS) reporter gene by histochemical and fluorimetric methods showed that all three promoters drive enhanced expression of GUS specifically in anther tissues and in pollen grains. In at least two of these promoters the –200/–100 regions actively convey the pollen/anther-specific expression in gain of function experiments using CaMV 35S as a minimal promoter. These nCI promoters thus contain a specific regulatory region responding to the physiological demands on mitochondrial function during pollen maturation. Pollen-specific motifs located in these regions appear to consist of as little as seven nucleotides in the respective promoter context.     

Pollen-specific expression of theArabidopsis thaliana α1-tubulin promoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genespromoter assayed by β-glucuronidase,chloramphenicol acetyltransferase and diphtheria toxin reporter genes

We have characterized the promoter specificity of theArabidopsis thaliana α₁-tubulin (α ₁-tub) gene by studying expression patterns of gene fusions between the 2.2 kbp 5′ upstream region of theα ₁-tub gene and each of three different reporters: chloramphenical acetyltransferase, β-glucuronidase or the diphtheria toxin chain A gene. Analysis of transgenic tobacco andArabidopsis plants carrying the transgene showed that the chloramphenicol acetyltransferase and β-glucuronidase activities were not detected in any vegetative or reproductive organs except mature pollen. Transgenic tobacco plants carrying the diphtheria toxin chain A gene under the control of theα ₁-tub promoter were of normal phenotype but seed fertility was drastically reduced. Furthermore, the transgene could not be transmitted to the next generation through pollen, supporting the observation that theα ₁-tub promoter is active only in pollen. It was observed that the promoter activity was most active in mature pollen and decreased significantly duringin vitro pollen germination, indicating that the promoter is inactive or subdued in germinating pollen. The promoter activity was not affected by various plant growth hormones during pollen maturation.     

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     

The pea <Emphasis Type="Italic">PsEND1</Emphasis> promoter drives the expression of GUS in transgenic wheat at the binucleate microspore stage and during pollen tube development

Many plant genetic engineering applications require spatial expression of genes which in turn depends upon the availability of specific promoters. In cereals, genetic modification of flowering and grain setting to influence yield and grain quality is of significant interest. PsEND1 is a pea promoter that displays expression in the epidermis, connective tissue, endothecium and middle layers during different stages of anther development. No homeologous sequence of this promoter or its coding sequence has been found in cereals. This present work aimed at the characterization of the pea PsEND1 promoter driving the expression of the gusA gene in transgenic wheat. Nine transgenic lines were produced by particle bombardment and analyzed for the expression of the gusA gene throughout development by histochemical GUS staining and by RT-PCR in vegetative and reproductive tissues and organs. Expression of the gusA gene was first detected during pollen development, in microspores at binucleate stage. Activity of the gusA gene was also found in mature pollen, after anthesis. Following pollen grain germination, expression of the gusA gene was seen from an early stage of pollen tube formation until advanced stages, approaching the ovary. No further expression of the gusA gene was detected after fertilization, nor during seed development. The results reported here show that the PsEND1 promoter is functional in wheat and its patterns of expression may be of interest for the application of genetic modification in wheat breeding.     

Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature-and ABA-inducible genes,direct strong β-glucuronidase expression in guard cells,pollen and young developing seeds

The ability of most higher plants to withstand freezing can be enhanced by cold acclimation, although the freezing tolerance of plant tissues is also affected by their developmental stage. In addition, low temperature has pleiotropic effects on many plant developmental processes such as vernalization. The interaction between plant development and low temperature implies that some genes are regulated by both environmental factors and developmental cues. Although a number of cold-inducible genes from plants have been identified, information concerning their regulation during plant development is limited. In order to understand their developmental regulation and obtain possible clues as to function, the promoters of kin1 and cor6.6, two cold- and abscisic acid (ABA)-regulated genes from Arabidopsis thaliana, were fused to the -glucuronidase (GUS)-coding sequence and the resulting constructs were used to transform tobacco and A. thaliana. Transgenic plants with either the kin1 or cor6.6 promoter showed strong GUS expression in pollen, developing seeds, trichomes and, most interestingly, in guard cells. During pollen development, maximum GUS activity was found in mature pollen. In contrast, the maximum GUS activity during seed development was during early embryogenesis. These patterns of expression distinguish kin1 and cor6.6 from related lea genes which are strongly expressed during late embryogenesis. There was no major qualitative difference in patterns of GUS expression between kin1 and cor6.6 promoters and the results were similar for transgenic tobacco and Arabidopsis. Considering the results described, as well as those in an accompanying paper Wang et al., 1995, Plant Mol Biol 28: 605–617 (this issue), we suggest that osmotic potential might be a major factor in regulating the expression of kin1 and cor6.6 during several developmental processes. The implication of the results for possible function of the gene products is discussed.