Biotechnology requires robust and predictable expression of transgenes. Most commercial Genetically Modified (GM) crops contain the viral 35S promoter to drive insecticide and herbicide resistance genes. In cotton there have been reductions in efficacy of Bacillus thuringiensis toxin (Bt) expressing plants late in the season that have been attributed to reductions in promoter activity. We have used genomic approaches to identify cotton genes whose expression remains high during the season to find promoters that might better maintain expression of transgenes in the field. A cDNA library from young late season leaves was used to generate about 2000 ESTs. Clustering of ESTs was used to determine relative transcript abundance and identify the most highly-expressed genes. These were primarily photosynthetic and housekeeping genes and some metabolic genes. The ESTs were printed to a small cDNA microarray and probed with both early- and late-season leaf mRNAs. Absolute fluorescence levels were used to rank genes and confirm the EST abundance data. Candidate genes, including the small subunit of Rubisco (RbcS) were selected. An RbcS promoter (Genbank Accession DQ648074) was isolated and analysed in both Arabidopsis and cotton linked to a GUS reporter gene. Expression of the reporter gene was consistently high in green tissues throughout the life cycle of cotton in the glasshouse, and in the field. A number of other candidate promoters have been identified that may be useful in a variety of biotechnology applications.
Variable levels of transgene expression are frequently observed among independent transgenic plants. Although different chromatin condensation states surrounding randomly integrated transgenes have previously been thought to be one of the causes for this variability, condensation states of plant chromatin have not been examined systematically. In this study, to analyze the condensation states of Arabidopsis chromatin, we evaluated the degree of chromatin condensation by measuring overall accessibility to DNase I (DNase I sensitivity) at 500-base resolution. We analyzed 30 variably expressed genes in an 80-kb genomic region, a gene repressed by polycomb group and heterochromatin protein 1-like genes, two genes near the heterochromatin, and a retrotransposon within the genetically-defined centromere in Arabidopsis. The centromeric region was significantly DNase I insensitive, however, sensitivity of these genes was similar irrespective of the individual gene expression levels.
This paper describes an efficient evaluation system of Arabidopsis mutants for defense responses. Young A. thaliana seedlings were analyzed for the induction of reactive oxygen species (ROS) as well as defense gene expression by chitin oligosaccharide elicitor in microtiter plates. Combined with the analysis of ROS in microtiter plates, the system enabled to evaluate thousands of mutant plants within a couple of weeks. Pharmacological studies could be applied and provided information about the molecular machinery involved in the defense responses. Experiments with the dissected organs showed that the roots were responsible for most ROS generation while the gene activation was observed in all the organs. An example of the successful application of this system for the screening of Arabidopsis muntats is also presented. Thus, the system provides a promising approach to screen, or evaluate, novel mutants for a confined defense signaling cascade downstream of a specific elicitor.
Plants contain a regulatory pathway similar to that involving retinoblastoma (RB) protein in animals. Here, we analyze an RB homolog, tobacco RETINOBLASTOMA-RELATED1 (NtRBR1), that encodes a protein possessing 13 potential phosphorylation sites for cyclin-dependent kinases (CDKs). We found that 11 synthetic peptides containing sequences from NtRBR1 were phosphorylated differentially by imunoprecipitates of cyclin/CDK complexes extracted from tobacco BY-2 cells. We raised antibodies that specifically recognize these sites of phosphorylation in NtRBR1. Distinct sites were phosphorylated during the cell cycle, suggesting that NtRBR1 is phosphorylated by different types of cyclin/CDK complexes.
The Golgi membranes labeled with cis-Golgi marker GFP-SYP31 were isolated from suspension-cultured cells of rice transformed with 35S::GFP-SYP31 by floating through a discontinuous sucrose density gradient in the presence of 5 mM MgCl2. The specific fluorescence intensity of final membrane preparation increased to approximately 150-fold in comparison with that of the post-nucleus soluble fraction. Specific activity of membrane-bound α-mannosidase (cis-Golgi) markedly increased, but NDPase (medial-/trans-Golgi) and NADPH-cytochrome c reductase endoplasmic reticulum were weakly detected in the membrane fraction. The other organelle marker enzymes, cytochrome c oxidase (mitochondria), alkaline pyrophosphatase (plastid), and catalase (peroxisome) were not detectable. Comparative display of protein spots in GFP-SYP31-labeled membranes, microsomal membranes and soluble fraction on the two dimensional gels showed that some proteins are markedably concentrated in the cis-Golgi membrane fraction. Furthermore, the mass spectrometric analysis of proteins separated by SDS-polyacrylamide gel electrophoresis indicated that the highly purified Golgi membranes contained several membrane traffic-related proteins and ER resident proteins. These findings indicated a close relationship between the ER and the cis-Golgi membranes.
The cell line GTH4 generated from the F1 hybrid of Nicotiana gossei×N. tabacum reveals lethality at the seedling stage. The cells proliferated at 37°C but died at 26°C. GTH4S, a cell line derived from GTH4 that does not die at 26°C, was established. Differences in the genes and proteins expressed in the two cell lines were analyzed. GTH4 expressed the gene encoding ubiquitin-conjugating enzyme (E2) which was not expressed in GTH4S. PCR targeting this sequence indicated that GTH4S is missing a segment of DNA containing the E2 locus, suggesting that GTH4S is a deletion mutant of GTH4. An analysis of proteins using anion-exchange HPLC and SDS-PAGE revealed GTH4 to contain protein species not found in GTH4S. The genes and/or proteins involved in the expression of hybrid lethality can be analyzed by comparing these two cell lines.
Hop bitter resins and essential oils are biosynthesized and accumulated in lupulin glands of hop (Humulus lupulus L.). Valerophenone synthase (VPS) is involved in the first steps of biosynthesis of bitter resins, and the VPS gene is specifically expressed in lupulin glands on bracteoles and leaves. In this study, spatial and temporal expression of the VPS gene was studied using in-situ hybridization. No gene signals were observed in the early stage of lupulin gland development, but strong expression was observed when the cuticle was slightly detached from the glandular head cells. Expression levels were remarkably reduced with accumulation of hop bitter resins and essential oils in the sub-cuticular space. The findings suggest that development of lupulin glands is strictly divided into a growth phase and biosynthetic-secretory phase.
The bZIP transcription factors are involved in various aspects of plant development. Studies of bZIP group I genes in several species have indicated that they may play a role in vascular development. In order to elucidate the functions of Arabidopsis bZIP group I genes in vascular development, the expression pattern of seven AtbZIP group I genes, AtbZIP18, 29, 30, 51, 52, 59, and 69, were examined in relation to vascular development using promoter::reporter lines of transgenic Arabidopsis plants. AtbZIP18, 51, 52, and 59 were preferentially expressed in developing vascular cells including differentiating vessels and their precursor cells. AtbZIP18, 52, and 59 showed partially overlapping expression in vascular cells of cotyledons, and partially overlapping expression of AtbZIP51, 52, and 59 was observed in root vascular cells. These results suggest that th ese genes may have partially redundant functions in vascular development.
To perform comparative sequence and transcriptome analyses between Brassica rapa and Arabidopsis thaliana, we prepared a B. rapa cDNA microarray using 1,820 (ca. 2 K) cDNA clones selected from 2,166 non-redundant sequences of cDNA library of Chinese cabbage. The gene expression during infection with fungal pathogen Colletotrichum higginsianum and treatments with signaling molecules was analyzed using 2 K B. rapa and 1.2 K Arabidopsis cDNA microarrays. In B. rapa, the results suggested a large correlation coefficient between compatible pathogen C. higginsianum-infection and the treatment with salicylic acid, methyl jasmonate, or ethephon. The expression profiles of 145 counterpart gene sets between the B. rapa and Arabidopsis were distributed in the self-organizing map analysis. The 28% of them indicated similarities in the two species transcriptome. These expressed sequence tag (EST) and microarray data should provide a valuable resource for functional genomics on the crops.
Leguminous plants have a unique pathway for production of 5-deoxy-type flavonoids and isoflavonoids that is distinct from the general flavonoid pathways. 5-Deoxy(iso)flavonoids are believed to play important eco-physiological roles as antimicrobial compounds and symbiotic signals toward rhizobia. The branching point of the 5-deoxyflavonoid pathway is the formation of the deoxy-type chalcone (isoliquiritigenin), which is catalyzed by the co-action of chalcone synthase and polyketide reductase (PKR). In the course of the comparative genomics of legume-specific genes, we cloned a putative cDNA for PKR (cPKR1) of a model legume Lotus japonicus (Regel) K. Larsen. Genomic Southern analysis showed that L. japonicus has a gene family composed of two to four paralogous PKR genes. The overexpression of cPKR1 in a red-flowered cultivar of petunia, “Polo Red Target”, reduced anthocyanin accumulation and caused the formation of isoliquiritigenin and its putative derivatives. These results suggested that PKR1 encodes a PKR that functions in planta.
The fluorescent signal of GFP fused to CIG-B, which is one of the three DREB1/CBF (dehydration responsive element binding protein 1/C-repeat binding factor) homologs isolated from sweet cherry, was observed only in nucleus. The expression of GFP driven by CIG-B promoter was up-regulated by low-temperature. These results suggested that nuclear localization signal (NLS) motif in the N-terminal region of CIG-B protein and inducer of CBF expression (ICE)-like motifs in CIG-B promoter could be responsible for nuclear targeting and low temperature perception, respectively.
DNA isolation followed by random amplified polymorphic DNA (RAPD) analysis was assessed as a possible method to distinguish three Indonesia species of Brugmansia because these species cannot be distinguished by metabolite analysis or morphological observation when they are not flowering. The DNA fragments were obtained by PCR, using random primers of isolated DNA, and the RAPD analysis showed that each species had different DNA fragment patterns, thus allowing each species to be individually distinguishable. The DNA patterns of each species remained the same, regardless of the samples region in Indonesia.
The purpose of this study was to develop a reproducible efficient procedure for the transformation of Javanica rice cultivars from Indonesia. Five rice cultivars cultivated in Indonesia now were examined for their capacity on the callus growth, plant regeneration and transformation. Their potential was affected by genotype and medium. Regarding the quality of callus, type I calli produced higher plant regeneration frequency than type II calli. So type I calli were inoculated with A. tumefaciens harboring binary plasmid pAFT14, which had a hygromycin resistance (hpt) gene and a gus gene. In this study, we examined two media for two steps in the tissue culture process for transformation, i.e. callus inducing and plant regeneration. The results show that C-modified medium was the suitable media for callus induction in the most Javanica rice cultivars. Agrobacterium-mediated transformation system has been extended to five Javanica rice cultivars. Among them, Rojolele consistently gave the best performance.
Protoplast culture and shoot regeneration from protoplast-derived calli were compared among different organs of Solanum integrifolium and three of its wild relatives (S. abutiloides, S. scabrum, and S. toxicarium). Leaves, cotyledons, and hypocotyls were used as sources of protoplast preparation. After one month of culture, a high frequency of visible colony formation was obtained from cotyledon protoplasts of S. integrifolium and S. scabrum, hypocotyl protoplasts of S. integrifolium and S. abutiloides, and leaf protoplasts of S. integrifolium and S. toxicarium. In addition, when the primary culture was started at a density of 2.5−5×104 protoplasts ml−1, the highest frequency of colony formation was obtained. Moreover, when the colonies were subcultured for 7 days on solid callus-proliferation medium before being transferred to shoot-induction medium, the plant regeneration frequency improved to between 91.8 and 98.8%.
Southern hybridization-based zygosity analysis was done in a transgenic rice plant (Oryza sativa L. cv Pusa Basmati 1) generated by Agrobacterium-mediated transformation with a rice chitinase (chi11) gene. A T0 plant with two unlinked T-DNA insertions (A and A′), was chosen for the application of Southern hybridization analysis to study genetic separation of the two loci by segregation and to identify the homozygous and hemizygous plants in T1- generation. The T1 plants showed differences in band intensities that reflected the homozygous and hemizygous status of each of the two integration events. The predictions of zygosity of T1 plants were confirmed by analyzing segregation in T2 plants. Southern hybridization analysis is demonstrated as a simple and effective method to distinguish hemizygous and homozygous plants in the T1 generation itself.
The nuclear matrix attachment region (MAR) located upstream from the tobacco gene CHN50, designated CHN50MAR, consists of two MAR elements: S/M I and S/M II. In a direct gene transfer system using tobacco BY-2 cells, CHN50MAR produced a several-fold increase in colony formation of hygromycin-resistant (hygR) cells when arranged adjacent to both the 5′ and 3′ termini of a hygromycin-selective marker gene. S/M I and S/M II enhanced hygR colony formation to the same degree as CHN50MAR, suggesting that these MAR elements are functionally redundant for enhancing transformation. The presence of the marker genes in the hygR cells was confirmed using the polymerase chain reaction. These results imply that CHN50MAR and its MAR elements are available as transformation enhancers in direct gene transfer systems for plants.
Recently, the development of techniques for generating living modified organisms (LMOs) has become a vital issue worldwide, in both developing and developed countries. Movement of LMOs across boundaries and the uses of LMOs pose global biosafety issues. Trans-boundary movement is regulated internationally by the Cartagena Protocol on Biosafety (CPB) to the Convention on Biological Diversity (CBD), which seeks to protect the biological diversity of natural ecosystems from risks posed by the deliberate release of LMOs into the environment. The regulatory frameworks of individual nations also play an important role in biosafety rules and biotechnology development, especially in the process from research and development to commercialization. However, a number of countries have not yet implemented the CPB CBD. In particular, Taiwan has not yet developed a complete and workable regulatory framework. The information presented in this comparative study should be useful for stakeholders in Taiwan, and will illustrate modalities and specific issues that will be useful for countries that are in the process of developing a biosafety regulatory framework. These countries may integrate Japanese primary experiences and feedback for mutually elaborative regional systems.