Characterization of a pollen-preferential gene, BAN102, from Chinese cabbage

We isolated and characterized a pollen-preferential gene, BAN102, from Chinese cabbage and analyzed the activity of its promoter. There were three or four copies of the BAN102 gene in the Chinese cabbage genome that specifically expressed in pollen and pollen tube. There were 2137 bp of BAN102 genomic clone comprising 186 bp of protein coding region, and 1178 bp of 5′ and 773 bp of 3′ non-coding regions. TATA box were located at 1071 nt of the promoter region while the polyadenylation signal and polyadenylation site were at 1470 and 1486 nt of the 3′ non-coding region. BLAST search of BAN102 sequence showed that coding region of BAN102 gene was the greatest percent similarity with arabinogalactan protein (AGP23) gene from Arabidopsis thaliana. Promoter analysis using GUS gene as a reporter showed that the pollen-specificity of BAN102 resided within the −112 to −44 bp of proximal promoter from the transient expression in tobacco and Chinese cabbage plants.     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

Expression of a polyubiquitin promoter isolated from Gladiolus

A polyubiquitin promoter (GUBQ1) including its 5′UTR and intron was isolated from the floral monocot Gladiolus because high levels of expression could not be obtained using publicly available promoters isolated from either cereals or dicots. Sequencing of the promoter revealed highly conserved 5′ and 3′ intron splicing sites for the 1.234 kb intron. The coding sequence of the first two ubiquitin genes showed the highest homology (87 and 86%, respectively) to the ubiquitin genes of Nicotiana tabacum and Oryza sativa RUBQ2. Transient expression following gene gun bombardment showed that relative levels of GUS activity with the GUBQ1 promoter were comparable to the CaMV 35S promoter in gladiolus, tobacco, rose, rice, and the floral monocot freesia. The highest levels of GUS expression with GUBQ1 were attained with Gladiolus. The full-length GUBQ1 promoter including 5′UTR and intron were necessary for maximum GUS expression in Gladiolus. The relative GUS activity for the promoter only was 9%, and the activity for the promoter with 5′UTR and 399 bp of the full-length 1.234 kb intron was 41%. Arabidopsis plants transformed with uidA under GUBQ1 showed moderate GUS expression throughout young leaves and in the vasculature of older leaves. The highest levels of transient GUS expression in Gladiolus have been achieved using the GUBQ1 promoter. This promoter should be useful for genetic engineering of disease resistance in Gladiolus, rose, and freesia, where high levels of gene expression are important.     

The mannopine synthase promoter contains vectorial cis-regulatory elements that act as enhancers and silencers

A 479-bp bi-directional promoter controls the expression of two genes (mas1′ and mas2′) that encode enzymes for the synthesis of the opine mannopine in plant tissues infected with Agrobacterium tumefaciens. This 5′ regulatory region (mas promoter) contains all the cis-acting elements involved in mediating the complex regulatory properties of these genes in plants. Using different mas promoter regions fused to a minimal 35S promoter (35SΔ108), we found that the regulatory properties of these divergent promoters result from the presence of orientation-dependent negative and positive regulatory regions. Some of these elements have the unusual property of acting as enhancers in one orientation and as silencers in the other. Using electrophoretic mobility shift analysis (EMSA), we showed that the functional mas promoter regions identified by fluorometric and histochemical assays for reporter gene activity in transgenic plants have the ability specifically to bind nuclear protein factors from Nicotiana tabacum, Phaseolus vulgaris, Solanum tuberosum, and Arabidopsis thaliana. Received: 7 May 1999 / Accepted: 5 August 1999     

Molecular cloning of a novel chimeric HMW glutenin subunit gene <Emphasis Type="Italic">1Dx5′</Emphasis> from a common wheat line W958

A novel chimeric high-molecular-weight (HMW) glutenin subunit gene from a new common wheat line W958 (2n = 6x = 42) was isolated and characterized. SDS–PAGE analysis revealed that this glutenin subunit has similar electrophoretic mobility to 1Dx5, so it was designated 1Dx5′. Genomic DNA from W958 was amplified and a 2,505-bp fragment was obtained. The 1Dx5′ subunit showed a chimeric primary structure of 1Dx5 and 1Dx2, with the 1Dx5 sequence in the 5′ and middle repetitive regions and the 1Dx2 sequence in the repetitive domain and 3′ region. MALDI-TOF-MS analysis demonstrated that 1Dx5′ had a molecular weight of 86815.1 Da, close to that of an x-type glutenin subunit. Secondary structure analysis showed that this subunit had six helixes and one strand, including four helixes in the repetitive domain which could enhance the dough properties. Additionally, the promoter of 1Dx5′ was obtained and showed the same sequence as 1Dx5 or 1Dx2 except for a few base conversions. The promoter analysis indicated that the cis-acting regulatory elements of 1Dx5′ were the same as those of 1Dx5 and/or 1Dx2. Previously, we have demonstrated that this novel glutenin subunit is associated with good bread-making quality and comprises a very large proportion of the F2 segregation population. Consequently, we suggest that the amino acid residue composition and the secondary structure of the subunit may contribute to the bread-making quality. In summary, the novel 1Dx5′ gene could have greater potential in wheat quality improvement.     

Analysis of <Emphasis Type="Italic">Acropora muricata</Emphasis> Calmodulin (<Emphasis Type="Italic">CaM</Emphasis>) Indicates That Scleractinian Corals Possess the Ancestral Exon/Intron Organization of the Eumetazoan <Emphasis Type="Italic">CaM</Emphasis> Gene

Calmodulin (CaM), belonging to the tropinin C (TnC) superfamily, is one of the calcium-binding proteins that are highly conserved in their protein and gene structure. Based on the structure comparison among published vertebrate and invertebrate CaM, it is proposed that the ancestral form of eumetazoan CaM genes should have five exons and four introns (four-intron hypothesis). In this study, we determined the gene structure of CaM in the coral Acropora muricata, an anthozoan cnidarian representing the basal position in animal evolution. A CaM clone was isolated from a cDNA library constructed from the spawned eggs of A. muricata. This clone was composed of 908 nucleotides, including 162 base pairs (bp) of 5′-untranslated region (UTR), 296 bp of 3′-UTR, and an open reading frame 450 bp in length. The deduced amino acid indicated that the Acropora CaM protein is identical to that of the actiniarian, Metridinium senile, and has four putative calcium-binding domains highly similar to those of other vertebrate or invertebrate CaMs. Southern blot analysis revealed that Acropora CaM is a putative single-copy gene in the nuclear genome. Genomic sequencing showed that Acropora CaM was composed of five exons and four introns, with intron II not corresponding to any region in the actiniarian CaM gene, which possesses only four exons and three introns. Our results highlight that the coral CaM gene isolated from A. muricata has four introns at the predicted positions of the early metazoan CaM gene organization, providing the first evidence from the basal eumetazoan phylum to support the four-intron hypothesis.     

Male Gametic Cell-specific Histone <Emphasis Type="Italic">gH2A</Emphasis> Gene of <Emphasis Type="Italic">Lilium longiflorum</Emphasis>: Genomic Structure and Promoter Activity in the Generative Cell

A genomic clone containing the gH2A gene, a histone variant specifically expressed in male gametic cells within the pollen of Lilium longiflorum, was isolated. Sequence analysis revealed that the coding region of the gene is interrupted by one intron, as is the case with the somatic type of plant histone H2A genes, suggesting derivation from the same ancestral gene containing one intron. In addition, a 2.8-kbp fragment of the 5′ upstream region of gH2A contained TATA and CAAT boxes, but neither a plant histone-specific regulatory DNA element nor vegetative cell-specific cis-elements were found. A histochemical study of stable transformants demonstrated that the 5′ upstream region of the gene can drive gene expression specifically in the generative cell of pollen; no activity was detectable in the vegetative cell or in other reproductive and vegetative tissues of transgenic Nicotiana tabacum. These results strongly suggest that the generative cell can direct specific gene expression, that this expression may be regulated by a putative male gametic factor, and that the gH2A promoter may therefore serve as a useful male gametic cell fate marker in angiosperms.     

Structural feature of sorghum chloroplast psbA gene and regulation effects of its 5'-noncoding region*

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli. Project supported by the Chinese National “863” and “973” Projects     

Steroid hormone responsiveness of a family of closely related mouse proviral elements

Regulation of the mouse sex-limited protein (Slp) gene is unusual in that hormone response is conferred by the 5′ LTR of an upstream inserted provirus, dubbed the imposon (imp1). In a search for additional genes whose regulation has been affected by retrotransposition events, we isolated two partial proviral elements by stringent screening of a mouse genomic library. One clone (imp2) contained a portion of the envelope gene and a 3′ LTR that was nearly identical to the 3′ LTR of imp1; this similarity extended to insertion into a B1 repetitive element. The second proviral clone (imp3) contained a 5′ LTR and associated coding sequences, but lacked its 3′ LTR; the LTR of imp3 differed by 12% from the imp1 sequence. To assess potential hormone response, proviral enhancer regions cloned into reporter vectors were tested in transfection. The imp2 enhancer was similar in behavior to imp1, conferring both androgen and glucocorticoid induction in one fragment context and an androgen-specific response in another. In contrast, the imp3 enhancer allowed high expression in the absence of hormone and was less responsive to steroids in general and androgen in particular. These three proviral elements define a small family of steroid responsive proviruses in the mouse genome, and at least one member has had a lasting impact on an endogenous gene's regulation. Received: 29 April 1997 / Accepted: 14 July 1997     

Authentic seed-specific activity of the <Emphasis Type="Italic">Perilla</Emphasis> oleosin 19 gene promoter in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds. Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression. Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both EGFP intensity and fluorometric GUS activity, respectively.     

Evaluation of seed storage-protein gene 5′ untranslated regions in enhancing gene expression in transgenic rice seed

5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.     

Structural feature of sorghum chloroplastpsbA gene and regulation effects of its 5′-noncoding region

Comparative analysis reveals remarkahle homology between the sequences of bothpsbA gene nucleotides and the inferred amino acids of sorghum, a C₄ plant, and those of rice, a C₃ plant. The 5′-noncoding region of sorghumpsbA gene contains the conservative promoter elements, “—35” element and “—10” element, like the prokaryote and the promoter element, TATA box, like the eukaryote. As compared with that of the rice, an extra sequence of 7 bp is found in the leader sequence of the mRNA in the former. Using anin vitro system, it has been demonstrated that protein factor exists in sorghum chloroplast protein extract which specifically hinds to the 5′-noncoding region ofpsbA gene. Measurement of the expression of luciferase shows a 2–5 time greater reaction of the expression plasmids pALqs which contain leader region of sorghumpsbA gene than that of the expression plasmids pALqr which contain leader region of ricepsbA gene inE. coli.     

Characterization of an Oxidative Stress Inducible Nonspecific Lipid Transfer Protein Coding cDNA and its Promoter from Drought Tolerant Plant <Emphasis Type="Italic">Prosopis juliflora</Emphasis>

Abiotic stress is the principal cause of crop failure worldwide. Prosopis juliflora is a hardy plant reported to be tolerant to drought, salinity, extremes of soil pH, and heavy metal stress. Here, we report the characterization of a cDNA clone for a putative nonspecific lipid transfer protein (Pj LTP1) found abundantly in a drought stressed leaf cDNA library of P. juliflora and its promoter. A multiple sequence analysis of Pj LTP1 with well-established and officially designated nonspecific lipid transfer proteins allergens revealed high similarity at the amino acid level. Northern analysis of Pj LTP1 in P. juliflora leaves under oxidative stress revealed steady upregulation at the time points analyzed. A 929-bp fragment was isolated from the 5′ end of Pj LTP1, and transient reporter gene expression studies revealed it to be a functional promoter. Several cis-acting elements previously reported to function in stress response were found in this promoter. The possible reasons for changes in gene expression during stress in relation to the host plant’s stress tolerance mechanisms are discussed.     

Inducible expression of the gene of Zinnia elegans coding for extracellular ribonuclease in Nicotiana tabacum plants

The gene of Zinnia elegans L. coding for S-like extracellular ribonuclease (ZRNase II) was used to produce transgenic tobacco plants with an increased ribonuclease activity. The protein-coding part of ZRNase II included the signal peptide sequence so the transgenic protein was located extracellularly. The cDNA of ZRNase II was cloned under the control of 2′-promoter of the mannopine synthase (MAS 2′) gene from Ti-plasmid of Agrobacterium tumefaciens. It was shown that the resultant transgenic plants had an increased ribonuclease activity of the crude extracts and the induction of MAS 2′ promoter by wounding additionally increased the activity. The plants of two transforming lines characterized by different ribonuclease activities were used to analyze the transgene influence on plant resistance to tobacco mosaic virus. The plants demonstrated either absence of disease symptoms or a significant delay in their appearance, depending on the virus content in the inoculum and ribonuclease activity.