Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

Comparative functional analysis of three abiotic stress-inducible promoters in transgenic rice

To identify minimal effective promoters for driving abiotic stress-inducible transgene expression in rice, we selected promoter elements of three stress-responsive genes, viz. rab16A coding for dehydrin, OsABA2 coding for zeaxanthin epoxidase, and a gene coding for a hypothetical protein (HP1) based on the presence of ABA-, salt- and drought-responsive cis-acting elements. These were translationally fused to the gusA reporter gene and introduced into rice to study their effect on heterologous gene expression. The OsABA2 promoter was found to be the most effective and desirable promoter among the three in terms of driving a low constitutive transgene expression under normal conditions and high induction in response to ABA, salt and drought stress, the highest being a 12-fold induction in response to ABA. The rab16A and HP1 promoters resulted in high levels of constitutive expression. While induction of GUS activity was generally two- to threefold for all the treatments in roots for both the promoters, induction in leaves was generally insignificant, the exceptions being rab16A in response to continuous salt stress and HP1 in response to water deficit. It was also observed that the three promoters, in general, resulted in lower constitutive expression, but higher induction in roots as compared to leaves. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. R. Wu: Deceased. This paper is humbly dedicated to the memory of Professor Ray Wu.     

Antibodies raised against yeast HSP 104 cross-react with a heat-and abscisic acid-regulated polypeptide in rice

Antibodies raised against yeast heat shock protein (HSP) 104 recognized a heat-inducible polypeptide with a molecular mass of 110 kDa in shoot tissue of young rice seedlings. Root tissue of the same age showed no immuno-reaction with yeast HSP 104 antibodies. The 110 kDa polypeptide of rice was also shown to be abscisic acid-inducible in young seedlings. Though this polypeptide was seen to be constitutively present in the flag leaf of 90-day-old field-grown plant, it was not much affected by either heat shock or abscisic acid in this case.     

Expression pattern and activity of six glutelin gene promoters in transgenic rice

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with beta-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.     

Development of a transgenic hairy root system in jute (Corchorus capsularis L.) with gusA reporter gene through Agrobacterium rhizogenes mediated co-transformation

Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome, indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future as explants for plantlet regeneration to obtain stable transgenic jute plants.     

Evaluation of tissue specificity and expression strength of rice seed component gene promoters in transgenic rice

Using stable transgenic rice plants, the promoters of 15 genes expressed in rice seed were analysed for their spatial and temporal expression pattern and their potential to promote the expression of recombinant proteins in seeds. The 15 genes included 10 seed storage protein genes and five genes for enzymes involved in carbohydrate and nitrogen metabolism. The promoters for the glutelins and the 13 kDa and 16 kDa prolamins directed endosperm-specific expression, especially in the outer portion (peripheral region) of the endosperm, whilst the embryo globulin and 18 kDa oleosin promoters directed expression in the embryo and aleurone layer. Fusion of the GUS gene to the 26 kDa globulin promoter resulted in expression in the inner starchy endosperm tissue. It should be noted that the 10 kDa prolamin gene was the only one tested that required both the 5' and 3' flanking regions for intrinsic endosperm-specific expression. The promoters from the pyruvate orthophosphate dikinase (PPDK) and ADP-glucose pyrophosphorylase (AGPase) small subunit genes were active not only in the seed, but also in the phloem of vegetative tissues. Within the seed, the expression from these two promoters differed in that the PPDK gene was only expressed in the endosperm, whereas the AGPase small subunit gene was expressed throughout the seed. The GUS reporter gene fused to the alanine aminotransferase (AlaAT) promoter was expressed in the inner portion of the starchy endosperm, whilst the starch branching enzyme (SBE1) and the glutamate synthase (GOGAT) genes were mainly expressed in the scutellum (between the endosperm and embryo). When promoter activities were examined during seed maturation, the glutelin GluB-4, 26 kDa globulin and 10 kDa and 16 kDa prolamin promoters exhibited much higher activities than the others. The seed promoters analysed here exhibited a wide variety of activities and expression patterns, thus providing many choices suitable for various applications in plant biotechnology.     

Expression of betaine aldehyde dehydrogenase gene and salinity tolerance in rice transgenic plants

Betaine as one of osmolytes plays an important role in osmoregulation of most high plants. Betaine aldehyde dehydrogenase C BADH) is the second enzyme involved in betaine biosynthesis. The BADH gene from a halophite, Atriplex hortensis, was transformed into rice cultivars by bombarment method. Totally 192 transgenic rice plants were obtained and most of them had higher salt tolerance than controls. Among transgenic plants transplanted in the saline pool containing 0.5% NaCl in a greenhouse, 22 survived, 13 of which set seeds, and the frequency of seed setting was very low, only 10% . But the controls could not grow under the same condition. The results of BADH ac-tivity assay and Northern blot showed that the BADH gene was integrated into chromosomes of transgenic plants and expressed.     

Directed chromosomal integration and expression of the reporter gene gusA3 in Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a probiotic microbe that survives passage through the human gastrointestinal tract and interacts with the host epithelium and mucosal immune cells. The potential for L. acidophilus to express antigens at mucosal surfaces has been investigated with various antigens and plasmid expression vectors. Plasmid instability and antibiotic selection complicate the possibility of testing these constructs in human clinical trials. Integrating antigen encoding genes into the chromosome for expression is expected to eliminate selection requirements and provide genetic stability. In this work, a reporter gene encoding a β-glucuronidase (GusA3) was integrated into four intergenic chromosomal locations. The integrants were tested for genetic stability and GusA3 activity. Two locations were selected for insertion downstream of constitutively highly expressed genes, one downstream of slpA (LBA0169), encoding a highly expressed surface-layer protein, and one downstream of phosphopyruvate hydratase (LBA0889), a highly expressed gene with homologs in other lactic acid bacteria. An inducible location was selected downstream of lacZ (LBA1462), encoding a β-galactosidase. A fourth location was selected in a low-expression region. The expression of gusA3 was evaluated from each location by measuring GusA3 activity on 4-methyl-umbelliferyl-β-d-glucuronide (MUG). GusA3 activity from both highly expressed loci was more than three logs higher than the gusA3-negative parent, L. acidophilus NCK1909. GusA3 activity from the lacZ locus was one log higher in cells grown in lactose than in glucose. The differences in expression levels between integration locations highlights the importance of rational targeting with gene cassettes intended for chromosomal expression.