David McElroy

Construction of expression vectors based on the rice actin 1 (Act1) 5' region for use in monocot transformation.

SummaryIt has been previously reported that the 5′ region of the rice actin 1 gene (Act1) promoted high-level expression of a β-glucuronidase reporter gene (Gus) in transformed rice cells. In this paper we describe the construction of Act1-based expression vectors for use in monocot transformation. As part of the development of these vectors, we have evaluated the influence of the Act1 first intron, the Act1-Gus junction-encoded N-terminal amino acids, and the sequence context surrounding the Act1 and Gus translation initiation site on Act1-Gus gene expression in rice and maize cells. We have found that addition of Act1 intron 1 to the transcription unit of a Gus reporter gene under control of the cauli-flower mosaic virus (CaMV) 35S promoter stimulated GUS activity more than 10-fold in transformed rice cells. Optimization of the sequence context around the Gus translation initiation site resulted in a 4-fold stimulation of Gus expression in transformed rice cells. By utilizing both the Act1 intron 1 and optimized Gus translation initiation site, a 40-fold stimulation in Gus expression from the CaMV 35S promoter has been achieved in transformed rice cells; very similar results were obtained in transformed maize cells. Taken together these results suggest that the Act1-based expression vectors described here should promote the expression of foreign genes in most, if not all, transformed monocot cells to levels that have not previoulsy been attainable with alternative expression vectors.

Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests.

The gene encoding a cowpea trypsin inhibitor (CpTI), which confers insect resistance in trangenic tobacco, was introduced into rice. Expression of the CpTi gene driven by the constitutively active promoter of the rice actin 1 gene (Act1) leads to high-level accumulation of the CpTI protein in transgenic rice plants. Protein extracts from transgenic rice plants exhibit a strong inhibitory activity against bovine trypsin, suggesting that the proteinase inhibitor produced in transgenic rice is functionally active. Small-scale field tests showed that the transgenic rice plants expressing the CpTi gene had significantly increased resistance to two species of rice stem borers, which are major rice insect pests. Our results suggest that the cowpea trypsin inhibitor may be useful for the control of rice insect pests.

Systemic induction of a potato pin2 promoter by wounding, methyl iasmonate, and abscisic acid in transgenic rice plants

To address the question whether common signal(s) and transduction pathways are used to mediate a systemic wound response in monocot and dicot plants, a fusion of the potato proteinase inhibitor II gene (pin2) promoter and the bacterial β-glucuronidase gene (Gus)-coding region was introduced into rice. In transgenic rice plants, the expression of the pin2-Gus fusion gene displays a systemic wound response, although the expression level is relatively low. Incorporation of the first intron from the rice actin 1 gene (Act1) into the 5′-untranslated region of the pin2-Gus construct results in high-level, systemically wound-inducible expression of the modified construct in transgenic rice plants. Histochemical analysis shows that this high-level, wound-inducible expression is associated with the vascular tissue in both leaves and roots. Furthermore, the expression of the pin2-Act1 intron-Gus fusion gene in transgenic rice plants can be systemically induced by both methyl jasmonate (MJ) and the phytohormone abscisic acid (ABA). These results suggest that the signal(s) mediating the observed systemic wound response and certain steps of the transduction pathways are conserved between dicot and monocot plants. Transient expression assays show that the pin2-Act1 intron-Gus construct is also actively expressed in transformed cells and tissues of several other monocot plants. Thus, the wound-inducible pin2 promoter in combination with the rice Act1 intron 1 might be used as an efficient regulator for foreign gene expression in transgenic monocot plants.

Light-Regulated and Cell-Specific Expression of Tomato rbcS-gusA and Rice rbcS-gusA Fusion Genes in Transgenic Rice

A previously isolated rice (Oryza sativa) rbcS gene was further characterized. This analysis revealed specific sequences in the 5[prime] regulatory region of the rice rbcS gene that are conserved in rbcS genes of other monocotyledonous species. In transgenic rice plants, we examined the expression of the [beta]-glucuronidase (gusA) reporter gene directed by the 2.8-kb promoter region of the rice rbcS gene. To examine differences in the regulation of monocotyledonous and dicotyledonous rbcS promoters, the activity of a tomato rbcS promoter was also investigated in transgenic rice plants. Our results indicated that both rice and tomato rbcS promoters confer mesophyll-specific expression of the gusA reporter gene in transgenic rice plants and that this expression is induced by light. However, the expression level of the rice rbcS-gusA gene was higher than that of the tomato rbcS-gusA gene, suggesting the presence of quantitative differences in the activity of these particular monocotyledonous and dicotyledonous rbcS promoters in transgenic rice. Histochemical analysis of rbcS-gusA gene expression showed that the observed light induction was only found in mesophyll cells. Furthermore, it was demonstrated that the light regulation of rice rbcS-gusA gene expression was primarily at the level of mRNA accumulation. We show that the rice rbcS gene promoter should be useful for expression of agronomically important genes for genetic engineering of monocotyledonous species.

Structural characterization of a rice actin gene

We have isolated and sequenced a full-length cDNA clone containing information for the rice actin gene RAc1. Transcript terminus mapping and sequence alignment between the RAc1 cDNA clone and a previously isolated RAc1 genomic clone were used to determine the structure of the RAc1 gene. This allowed us to make the first complete structural characterization of a plant actin gene. The analysis revealed the presence of a 5′-noncoding exon, separated by an intron, from the first translated exon of the RAc1 gene. This is one of the few reported cases of a plant gene containing such a 5′-noncoding exon. Sequence comparison between the previously isolated plant actin genes suggests that such an exon may be a common feature of plant actin gene structure. The present study also confirms that the rice actin gene family is composed of at least eight unique members.

Characterization of the rice (Oryza sativa) actin gene family.

Four rice (Oryza sativa) genomic actin genes have been characterized. The rice actin genes show a conservation of intron number and position that is characteristic of plant actins. Sequence comparisons revealed that the plant actins generally have a high degree of inter- and intraspecific sequence heterogeneity. However, one rice actin gene has a much higher degree of nucleotide sequence identity to a previously isolated actin sequence from Arabidopsis thaliana than to any other plant actin gene. This leads us to suggest that the two sequences may represent functionally homologous genes which arose from an ancient actin gene lineage that was separated by the divergence of the dicot and monocot plants. Genomic DNA blot analysis showed that the rice actin gene family contains at least eight unique members. RNA hybridization analysis revealed that individual rice actin genes can display different patterns of transcript accumulation. The observed differences in sequence and transcript accumulation patterns suggest that the individual rice actin genes may differ in their transcriptional regulation and/or cellular function.

Genomic nucleotide sequence of four rice (Oryza sativa) actin genes.

Cytoplasmic actin is a fundamental component of the eukaryotic cell cytoskeleton. In higher plant cells a number of cellular processes, such as cytoplasmic streaming, extension growth and cell division, are believed to involve cytoskeletal actin protein [ 8 ]. Actin has been found to be encoded by gene families in a number of different higher plants [ 1, 4, 6, 7]. As a first step towards a characterization of the actin genes in rice we have isolated several clones from a rice genomic library representing four unique actin sequences. A rice (Oryza sativa cv. IR26) genomic library in 2EMBL4 (Y. Xie and R. Wu, unpublished results) was screened with a heterologous actin probe. Fourteen independent clones were isolated and suncloned into pUC 13. By mapping restriction sites and carrying out cross-hybridization studies, four different classes of clones were identified and designated RAcl, RAc2, RAc3and RAc7. Sequencing of the subcloned actin genes was carried out by the dideoxynucleotide chain-termination method and computer analysis of the resulting sequences was done using the Microgenie sequence analysis program [ 5 ]. The nucleotide sequences of the coding regions of the four actin genes are presented in Fig. 1. A sequence alignment between a 1.5 kb RAcl cDNA clone [2] and the four genomic actin clones was made to determine the probable number and location of introns within the actin coding regions. This analysis identified three introns located in the same position in all four rice actin coding regions as that previously reported for the other known plant actin genes. In the majority of cases the predicted intron splice site junctions have expected structure of G : GT. . .AG : G at their borders [3 ]. The sequences of RAcl, RAc2, RAc3 and RAc7 (if translated in vivo) would code for 377, 377, 375 and 375 amino acides with estimated molecular masses of 41.7, 42.0, 41.8 and 41.7 kDa respectively. The coding sequences of the four rice actin genes were compared directly to each other to yield percentage identity at the nucleotide and amino acid levels. This analysis revealed that the rice actin genes. Like other plant actins [7], are highly diverged from each other at both the nucleotide and amino levels. The percent nucleotide

Characterization of cis-acting elements regulating transcription from the promoter of a constitutively active rice actin gene.

The promoter of the constitutively expressed rice (Oryza sativa) actin 1 gene (Act1) is highly active in transformed rice plants (W. Zhang, D. McElroy, and R. Wu, Plant Cell 3:1150-1160, 1991). A region 834 bp upstream of the Act1 transcription initiation site contains all the regulatory elements necessary for maximal gene expression in transformed rice protoplasts (D. McElroy, W. Zhang, J. Cao, and R. Wu, Plant Cell 2:163-171, 1990). We have constructed a series of Act1 promoter deletions fused to a bacterial beta-glucuronidase reporter sequence (Gus). Transient expression assays in transformed rice protoplasts, as well as transformed maize cells and tissues, identified two distinct cis-acting regulatory elements in the Act1 promoter. A 38-bp poly(dA-dT) region was found to be a positive regulator of Act1 promoter activity. Deletion of the poly(dA-dT) element lowered Gus expression by at least threefold compared with expression produced by the full-length Act1 promoter. By gel retardation and footprinting, we identified a ubiquitous rice protein which specifically recognizes this poly(dA-dT) element in the constitutively active Act1 promoter. A CCCAA pentamer repeat-containing region was found to be a negative regulator of the Act1 promoter in transformed rice protoplasts. Transient expression assays in different maize cells and tissues with use of the Act1 deletion constructs suggested that the CCCAA pentamer repeat region functions in a complex tissue-specific manner. A CCCAA-binding protein was detected only in root extracts.

Function and evolution of actins

In this review, evolutionary relationships of actin gene and protein sequences, as well as intron positions within actin genes, are analyzed both within and between eukaryotic kingdoms.

Effect of the cauliflower mosaic virus 35S, actin, and ubiquitin promoters on uidA expression from a Bar-uidA fusion gene in transgenic Gladiolus plants.

SummaryTissue-specific patterns and levels of gene expression were characterized in transgenic Gladiolus plants that contained the phosphinothricin acetyltransferase (bar)-β-glucuronidase (uidA) fusion gene under transcriptional control of the promoter from either the cauliflower mosaic virus 35S (CaMV 35S), duplicated CaMV 35S (2×CaMV 35S), rice actin (Act1), or Arabidopsis ubiquitin (UBQ3) promoters. The bar gene confers resistance to phosphinothricin (PPT)-containing herbicides and allowed selection of transgenic cells. The β-glucoronidase gene encoded by the uidA locus of E. coli functioned as a reporter gene. Maximum levels of β-glucuronidase (GUS) activity in leaves were 173, 112, 50, and 10 nmoles 4-methylumbelliferone h−1 mg−1 protein for transgenic plants with the bar-uidA fusion gene under the control of the CaMV 35S, 2×CaMV 35S, UBQ3, and Act1 promoters, respectively. There was frequently considerable variability in GUS activity between the leaves of a single plant, and levels of uidA expression varied between independently transformed plants for each promoter. Callus derived from transgenic plants showed much less variation in GUS expression than leaves. The mean level of GUS activity was significantly higher (over 3×) for transgenic lines of callus containing the CaMV 35S as compared to the UBQ3 promoter, and this confirmed the higher (2×) level of GUS activity in levels of plants with the CaMV 35S promoter as compared to the UBQ3 promoter. Tissue-specific patterns of uidA expression were determined by histochemical staining. Leaves 5–6 cm long from plants with any of the four promoters tested exhibited uidA expression primarily in the vasculature. Under all four promoters uidA was expressed more frequently in root tips as compared to leaves.