Matrix attachment region from the chicken lysozyme locus reduces variability in transgene expression and confers copy number-dependence in transgenic rice plants.

Matrix-attachment regions (MARs) may function as domain boundaries and partition chromosomes into independently regulated units. In this study, BP-MAR, a 1.3-kb upstream fragment of the 5'MAR flanking the chicken lysozyme locus, was tested for its effects on integration and expression of transgenes in transgenic rice plants. Using the Agrobacterium-mediated method, we transformed rice with nine different constructs containing seven and six different promoters and coding sequences, respectively. Genomic Southern blot analyses of 357 independent transgenic lines revealed that in the presence of BP-MAR, 57% of the lines contained a single copy of the transgene, whereas in its absence, only 20% of the lines contained a single copy of the transgene. RNA gel-blot and immunoblot experiments demonstrated that in the presence of BP-MAR, transgene expression levels were similar among different lines. These data were in direct contrast to those derived from transgenes expressed in the absence of BP-MAR, which varied markedly with the chromosomal integration site . Thus, it can be concluded that BP-MAR significantly reduces the variability in transgene expression between independent transformants. Moreover, the presence of BP-MAR appears to confer a copy number-dependent increase in transgene expression, although it does not increase expression levels of individual transgenes. These data contrast with results previously obtained with various MARs that increased expression levels of transgene significantly. Therefore, we conclude that the incorporation of BP-MAR sequences into the design of transformation vectors can minimize position effects and regulate transgene expression in a copy number-dependent way.

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweet potato and its expression in response to stress.

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1, was isolated from cell cultures of sweet potato ( Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweet potato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 microM), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 microM) or exposure to high temperature (37 degrees C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen ( Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.

Triprimer-PCR method: rapid and reliable detection of transgenes in transgenic rice plants.

We designed a triprimer-PCR system for detection of transgenes and applied for analysis of two different kinds of transgenic rice plants, which were previously transformed with the plasmids pBY605RR or pARP7 containing a maize ribosome-inactivating protein gene, Zmcrip3a, and a herbicide-resistant gene, bar. Genomic Southern-blot analysis demonstrated that the transgenes were stably inherited to their R1 progenies without changes in configuration. The resulting data were used as a reference for triprimer-PCR analysis. The triprimer-PCR system uses an endogenous gene as an internal standard which shares an identical priming site for one primer with a transgene while each of the other two primers is specific to either the transgene or the endogenous gene. Triprimer-PCR analysis was carried out on genomic DNA isolated from 24 different progenies of the pBY605RR- and the pARP7-transformed lines that contain different copy numbers of transgenes. The RbcS:Zmcrip3a junction region of the pBY605RR integrated in rice chromosomes, together with the endogenous RbcS, was efficiently amplified, producing 440 and 250 bp expected PCR products. Also, the Act1:Zmcrip3a junction region of the transgene pARP7 with the endogenous Act1 was similarly amplified, producing 540 and 340 bp expected PCR products. The two PCR products in each set of experiments were observed consistently and independently of copy numbers or rearrangements of the transgene. Thus, the triprimer-PCR strategy may provide a rapid and reliable method for confirming transformation or analyzing segregation of transgenes at the molecular level.