Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant ''resurrection plants, '' trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3-10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mg͞g fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.T he explosive increase in world population, along with the continuing deterioration of arable land, scarcity of fresh water, and increasing environmental stress pose serious threats to global agricultural production and food security. Despite focused efforts to improve major crops for resistance to abiotic stresses (1) such as drought, excessive salinity, and low temperature by traditional breeding, success has been limited. This lack of desirable progress is attributable to the fact that tolerance to abiotic stress is a complex trait that is influenced by coordinated and differential expression of a network of genes. Fortunately, it is now possible to use transgenic approaches to improve abiotic stress tolerance in agriculturally important crops with far fewer target traits than had been anticipated (2).Abiotic stresses can directly or indirectly affect the physiological status of an organism by altering its metabolism, growth, and development. A common response of organisms to drought, salinity, and low-temperature stresses is the accumulation of sugars and other compatible solutes (3). These compounds serve as osmoprotectants and, in some cases, stabilize biomolecules under stress conditions (3,4). One such compound is trehalose, a nonreducing disaccharid...

We have characterized the 5' region of the rice actin 1 gene (Act1) and show that it is an efficient promoter for regulating the constitutive expression of a foreign gene in transgenic rice. By constructing plasmids with 5' regions from the rice Act1 gene fused to the coding sequence of a gene encoding bacterial beta-glucuronidase, we demonstrate that a region 1.3 kilobases upstream of the Act1 translation initiation codon contains all of the 5'-regulatory elements necessary for high-level beta-glucuronidase (GUS) expression in transient assays of transformed rice protoplasts. The rice Act1 primary transcript has a noncoding exon separated by a 5' intron from the first coding exon. Fusions that lack this Act1 intron showed no detectable GUS activity in transient assays of transformed rice protoplasts. Deletion analysis of the Act1 5' intron suggests that the intron-mediated stimulation of GUS expression is associated, in part, with an in vivo requirement for efficient intron splicing.

We have characterized the 5' region of the rice actin 1 gene (Actl) and show that it is an efficient promoter for regulating the constitutive expression of a foreign gene in transgenic rice. By constructing plasmids with 5' regions from the rice Actl gene fused to the coding sequence of a gene encoding bacterial P-glucuronidase, we demonstrate Pthat a region 1.3 kilobases upstream of the Actl translation initiation codon contains all of the 5'-regulatory elements necessary for high-leve1 P-glucuronidase (GUS) expression in transient assays of transformed rice protoplasts. The rice Actl primary transcript has a noncoding exon separated by a 5' intron from the first coding exon. Fusions that lack this Actl intron showed no detectable GUS activity in transient assays of transformed rice protoplasts. Deletion analysis of the Act 7 5' intron suggests that the intron-mediated stimulation of GUS expression is associated, in part, with an in vivo requirement for efficient intron splicing.

Unequal sister chromatid exchanges occur at the ribosomal DNA locus of yeast during mitotic growth. The frequency of unequal crossing over, as measured by the deletion or duplication of an inserted genetic marker (LEU2), is sufficient to maintain the sequence homogeneity of the rDNA repeat units.

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We introduced the potato proteinase inhibitor II (PINII) gene (pin2) into several Japonica rice varieties, and regenerated a large number of transgenic rice plants. Wound-inducible expression of the pin2 gene driven by its own promoter, together with the first intron of the rice actin 1 gene (act1), resulted in high-level accumulation of the PINII protein in the transgenic plants. The introduced pin2 gene was stably inherited in the second, third, and fourth generations, as shown by molecular analyses. Based on data from the molecular analyses, several homozygous transgenic lines were obtained. Bioassay for insect resistance with the fifth-generation transgenic rice plants showed that transgenic rice plants had increased resistance to a major rice insect pest, pink stem borer (Sesamia inferens). Thus, introduction of an insecticidal proteinase inhibitor gene into cereal plants can be used as a general strategy for control of insect pests.

Several electrophoretic and chromatographic systems have been investigated and compared for sequence analysis of oligodeoxyribonucleotides. Three systems were found to be useful for the separation of a series of sequential degradation products resulting from a labeled oligonucleotide: (I) 2-D electrophoresisdagger; (II) 2-D PEI-cellulose; and (III) 2-D homochromatography. System (III) proved generally most informative regardless of base composition and sequence. Furthermore, only in this system will the omission of an oligonucleotide in a series of oligonucleotides be self-evident from the two-dimensional map. The sequence of up to fifteen nucleotides can be determined solely by the characteristic mobility shifts of its sequential degradation products distributed on the two-dimensional map. With this method, ten nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and seven from the right-hand 3'-terminus of bacteriophage lambda DNA have been sequenced. Similarly, nine nucleotides from the double-stranded region adjacent to the left-hand 3'-terminus and five nucleotides from the right-hand terminus of bacteriophage phi80 DNA have also been sequenced. The advantages and disadvantages of each separation system with respect to sequence analysis are discussed.

The complete DNA sequence of the human papovavirus BK is presented. From the 4963 base-pair sequence of BK virus (MM strain), the amino acid sequence of at least five proteins can be deduced: a T antigen and a t antigen, which share amino terminal peptides; proteins VP2 and VP3, which share 232 amino acids; and protein VP1, whose coding sequence overlaps those for VP2 and VP3 by 113 nucleotides but is read in a different frame. The gene loci and the arrangement of genes are strikingly similar in BK virus and simian virus 40 (SV40). The sequence of the deduced proteins in BK virus shares 73 percent amino acid homology with those in SV40, whereas the DNA sequence of the two viruses shares 70 percent homology, suggesting close evolutionary relationship. However, the repeated DNA sequences in the noncoding regions of these viruses are different.