Gene stability in transgenic aspen (Populus). I. Flanking DNA sequences and T-DNA structure

The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L.?×?tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-##1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions.     

Gene stability in transgenic aspen ( Populus ). I. Flanking DNA sequences and T-DNA structure

The stability of transgenes in the genome of transformed plants depends strongly on their correct physical integration into the host genome as well as on flanking target DNA sequences. For long-lived species like trees, however, no information is available so far concerning inactivation or loss of transgenes due to gene silencing or somatic genome rearrangement events. In this study, four independently transformed 35S-rolC transgenic hybrid aspen plants (Populus tremula L. × tremuloides Michx.), each harbouring one copy of the transgene, were investigated during continuous growth in the greenhouse. In one of these transgenic lines (Esch5:35S-rolC-##1) individuals frequently show phenotypic reversions, while in the remaining three lines (Esch5:35S-rolC-#3, -#5, -#16) the gene was essentially stable. Molecular analysis including PCR, Southern and Northern assays clearly showed that the transgene had been lost in the revertant tissue of the unstable line. Sequencing of T-DNA right and left borders, and flanking DNA regions, in all four transgenic aspen lines revealed no differences either in the type of flanking DNA (G-C to A-T ratio) or with respect to the presence of enhancers or MAR (matrix associated repeats)-like structures. Primers located within the left and right flanking regions in the three stable lines could be used to recover the target sites from the untransformed plants. This was not possible, however, with the unstable line, indicating that at least one flanking sequence does not derive from the plant target DNA but is of unknown origin. PCR using other primer pairs, and inverse PCR analysis, revealed an additional truncated T-DNA copy of 1050 nucleotides adjacent to the left border of the complete copy in this line. Sequencing of this truncated T-DNA revealed that it represented an inverted copy of part of the right half of the original construct. This special feature would allow the inverted repeat to pair with right border sequences of the complete copy. This would explain the frequently observed reversion resulting in transgene loss as due to intrachromosomal base-pairing leading to double-stranded loops of single-stranded DNA during mitotic cell divisions. Received: 9 June 1998 / Accepted: 6 October 1998     

Molecular characterization of T-DNA integration sites in transgenic birch

The integration and structure of a transgene locus can have profound effects on the level and stability of transgene expression. We screened 28 transgenic birch (Betula platyphylla Suk.) lines transformed with an insect-resistance gene (bgt) using Agrobacterium tumefaciens. Among the transgenic plants, the copy number of transgene varied from one to four. A rearrangement or partial deletion had occurred in the process of T-DNA integration. T-DNA repeat formation, detected by reverse primer PCR, was found among randomly screened transgenic lines. Sequencing of the junctions between the T-DNA inserts revealed deletions of 19–589 bp and an additional 45 bp filler DNA sequence was inserted between the T-DNA repeats at one junction. Micro-homologous sequences (1–6 bp) were observed in the junctions between the T-DNA inserts. Using SiteFinding-PCR, a relatively high percentage of AT value was found for the flanking regions. Deletion of the right border repeat was observed in 12/18 of the T-DNA/plant junctions analyzed. The number of nucleotides deleted varied from 3 to 712. Deletions of 17–89 bp were observed in all left T-DNA/plant junctions analyzed. A vector backbone DNA sequence in the transgene loci was also detected using primer pairs outside the left and right T-DNA borders. Approximately 89.3% of the lines contained some vector backbone DNA. These observations revealed that it is important to check the specificity of the integration. A mechanism of T-DNA transport and integration is proposed for this long-lived tree species.     

A simple,rapid and systematic method for the developed GM rice analysis

We generated 383 independent transgenic lines that contained the PsGPD (Glyceraldehyde-3-Phosphate Dehydrogenase), ArCspA (Cold Shock Protein), BrTSR15 (Triple Stress Resistance 15) and BrTSR53 (Triple Stress Resistance 53) genes under the control of a constitutive (CaMV 35S) promoter to generate genetically modified (GM) rice. TaqMan copy number assay was performed to determine the copy numbers of inserted T-DNA. Flanking sequence tags (FSTs) were isolated from 203 single copy T-DNA lines of transgenic plants, and their sequences were mapped to the rice chromosomes. Of the 157 flanking sequence tags that were isolated from single copy lines, transgenes were found to be integrated into genic regions in 58 lines (36 %), whereas 97 lines (62 %) contained transgene insertions in intergenic regions. Approximately 27 putative homozygous lines were obtained through multi-generations of planting, resistance screening and TaqMan copy number assays. To investigate the transgene expression patterns, quantitative real-time PCR analysis was performed using total RNA from leaf tissue of homozygous T1 plants with a single copy and an intergenic insertion of T-DNA. The mRNA expression levels of the examined transgenic rice were significantly increased in all transgenic plants. In addition, myc-tagged 35S:BrTSR15 and 35S:BrTSR53 transgenic plants displayed higher levels of transgene protein. Using numerical data for the mass production of transgenic plants can reduce the time required to obtain a genetically modified plant. Moreover, the duration, cost, and efforts required for transformation can be deliberately predicted. These results may be useful for the large-scale production of transgenic plants or T-DNA inserted rice mutants.     

Integration,expression and inheritance of two linked T-DNA marker genes in transgenic lettuce

T-DNA integration and stability were assessed in Agrobacterium-derived transgenic lettuce lines carrying a chimaeric CaMV 35S promoter-driven gus-intron gene and a chimaeric nos.nptII.nos gene. T-DNA integration was predominantly complex in transgenic plants derived from an A. tumefaciens strain carrying the supervirulent plasmid ToK47. Truncation of the right side of the T-DNA was observed in first seed generation R₁ plants from one line. Complex T-DNA integration patterns did not always correlate with low transgene expression. Despite a high T-DNA copy number, ca. 30% of the lines analysed showed high transgene expression in the R₁ generation. High transgene expression was stable at least to the R₄ seed generation in selected high-expressing lines. Transgene expression was lost in the R₂ generation in a low expressing line, while complete, heritable transgene silencing from the R₀ to R₂ generations was also observed in another line. A 50-fold variation in -glucuronidase (GUS) activity and a 16-fold variation in NPTII protein content were observed between R₁ plants derived from different R₀ parents. Reactivation of transgene expression with 5-azacytidine in partially silenced lines indicated that low expression was associated with DNA methylation.     

Comparative transcriptome analysis reveals whole-genome duplications and gene selection patterns in cultivated and wild <Emphasis Type="Italic">Chrysanthemum</Emphasis> species

Key message

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. T-DNA inserts are stable; no transgene rearrangements were observed. AmCYAN1 and PMI protein accumulation levels were maintained. There was no evidence that production of either protein declined across generations and no transgene silencing was observed in three commercial sugarcane varieties through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes over 4 years of field testing. Long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

Abstract

This study addresses T-DNA insert stability and transgene expression consistency in multiple cycles of field propagated sugarcane. These data are critical supporting information needed for successful commercialization of GM sugarcane. Here seventeen transgenic events, containing the AmCYAN1 gene driven by a CMP promoter and the E. coli PMI gene driven by either a CMP or Ubi promoter, were used to monitor T-DNA insert stability and consistency of transgene encoded protein accumulation through commercially relevant ratooning, propagation-by-setts, and micro-propagation generation processes. The experiments were conducted in three commercial sugarcane varieties over 4 years of field testing. DNA gel blot analysis showed that the T-DNA inserts are stable; no transgene rearrangements were observed. Quantitative ELISA showed no evidence of decreasing AmCYAN1 and PMI protein levels across generations and no transgene silencing was observed. These results indicate that long term transgene expression consistency and T-DNA insert stability can be achieved in sugarcane, suggesting that it is highly probable that transgenic sugarcane can be successfully commercialized.

     

A large-scale study of rice plants transformed with different T-DNAs provides new insights into locus composition and T-DNA linkage configurations

Transgenic locus composition and T-DNA linkage configuration were assessed in a population of rice plants transformed using the dual-binary vector system pGreen (T-DNA containing the bar and gus genes)/pSoup (T-DNA containing the aphIV and gfp genes). Transgene structure, expression and inheritance were analysed in 62 independently transformed plant lines and in around 4,000 progeny plants. The plant lines exhibited a wide variety of transgenic locus number and composition. The most frequent form of integration was where both T-DNAs integrated at the same locus (56% of loci). When single-type T-DNA integration occurred (44% of loci), pGreen T-DNA was preferentially integrated. In around half of the plant lines (52%), the T-DNAs integrated at two independent loci or more. In these plants, both mixed and single-type T-DNA integration often occurred concurrently at different loci during the transformation process. Non-intact T-DNAs were present in 70–78% of the plant lines causing 14–21% of the loci to contain only the mid to right border part of a T-DNA. In 53–66% of the loci, T-DNA integrated with vector backbone sequences. Comparison of transgene presence and expression in progeny plants showed that segregation of the transgene phenotype was not a reliable indicator of either transgene inheritance or T-DNA linkage, as only 60–80% of the transgenic loci were detected by the expression study. Co-expression (28% of lines) and backbone transfer (53–66% of loci) were generally a greater limitation to the production of marker-free T₁ plants expressing the gene of interest than co-transformation (71% of lines) and unlinked integration (44% of loci).     

Vegetative and generative dispersal capacity of field released transgenic aspen trees

Transfer of genes by pollen or wind-dispersed seed is considered a main potential risk when field release experiments with transgenic trees are initiated. In Germany, the first release experiment with genetically transformed trees was initiated in 1996. To ensure that the transgenic trees remained in the vegetative phase, the duration of the experiment was limited to 5 years. In total, 457 1-year-old trees including eight transgenic aspen lines carrying either the 35S- rolC or the rbcS- rolC gene construct, and three control clones were transferred to the field. In 1998 and 2000, 12 plants of transgenic lines all carrying the 35S- rolC gene construct formed female flower buds. Furthermore, one young aspen plant identified as a root sucker was observed in 1999 followed by an increasing number of root suckers derived from transgenic and non-transgenic trees in 2000 and 2001. In 2001, the last year of the field trial, 15 root suckers were detected outside the field. In total, 234 root suckers were harvested in 2000 and 2001 and analysed for their transgenic status. More than half of the roots suckers investigated showed the presence of the rbcS- rolC gene construct. We concluded that in addition to the widely accepted generative propagation, vegetative dispersal capacity of transgenic perennial plants is also important and must be included in risk assessment studies.     

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.     

A tapetal ablation transgene induces stable male sterility and slows field growth in Populus

The field performance of genetic containment technologies–considered important for certain uses of transgenic trees in forestry–is poorly known. We tested the efficiency of a barnase gene driven by the TA29 tapetum-dominant promoter for influencing growth rate and inducing male sterility in a field trial of transgenic hybrid poplar (Populus tremula?×?Populus tremuloides). When the growth of 18 transgenic insertion events with the sterility transgene were compared to non-transgenic controls after two growing seasons, they grew 40 % more slowly in stem volume, and all but one transgenic event grew significantly more slowly than the control. In contrast, when we compared the growth of transgenic trees containing four kinds of β-glucuronidase (GUS) reporter gene constructs to non-transgenic trees—all of which had been produced using the same transformation method and poplar clone and grown at the same field site—there were no statistically significant differences in growth after three growing seasons. In 2 years where gross pollen release from catkins was monitored and found to be abundant in the control, no pollen was visible in the transgenic trees; microscopy suggested the cause was tapetal collapse and revealed the presence of a very few normal-sized pollen grains of unknown viability. In two additional years when viable, well-formed pollen was microscopically documented in controls, and no pollen could be observed in any transgenic trees. We conclude that this construct resulted in robust and possibly complete male sterility that was stable over 4 years in the field.     

Enhanced drought and salt tolerance by expression of AtGSK1 gene in poplar

A GSK3/shaggy-like kinase (AtGSK1) has been implicated in the regulation of drought and salt tolerance. We transferred AtGSK1 from Arabidopsis thaliana to a hybrid poplar (Populus alba × P. tremula var. grandulosa) to determine the effect of the transgene expression in the transgenic trees. The results from northern blot and RT-PCR analyses showed that the expression level varied among the transgenic lines. During their culture on tissue culture media, the transgenic poplars formed vigorous growing roots even in the presence of 125 mM NaCl and callus in the presence of 150 mM NaCl. When the transgenic poplars were growing in pots and provided with NaCl solution, they stayed much healthier than did nontransgenic poplars, showing higher rates of photosynthetic rates, stomatal conductance, and evaporation rates under the stress. Whereas the total level of leaf Na⁺ level increased dramatically in transgenic poplars under severe saline conditions (150 mM NaCl), that of leaf K⁺ decreased in the same plants under the same conditions. Total root Na⁺ level increased in nontransgenic poplars under severe saline conditions. In contrast, total root K⁺ level decreased in the same plants under the same conditions. The chloride content and relative electrical conductivity of the transgenic poplars after salt stress treatment were lower than those of nontransgenic poplars. The transgenic poplars were also tolerant to up to 20 % PEG remaining significantly healthy when compared with nontransgenic poplars with necrosis and chlorosis symptoms. Another dramatic feature of the transgenic poplars was wilting tolerance for prolonged drought treatment up to 2 weeks. The results provide evidence that the expression of AtGSK1 gene conferred drought and salt tolerance in the transgenic poplars.     

The influence of the rolC gene on isoflavonoid production in callus cultures of Maackia amurensis

The polyphenolic complex of Maackia amurensis, as well as a complex of isoflavonoids from M. amurensis callus cultures, display strong hepatoprotective effects in experimental animal and human studies. To increase the yield of polyphenols in cultures of M. amurensis, calli were transformed with the rolC gene as well as with an empty vector that was used as a control. HPLC analysis revealed that the transgenic cultures produced the same complex of isoflavonoids. The complex consisted of 20 compounds, including isoflavones and their glucosides as well as pterocarpans and their glucosides. The cultures transformed with either the empty vector or the rolC gene construct produced on average 1.22 % dry weight (DW) and 1.39 % DW of isoflavonoids, respectively. Isoflavonoid production in the transformed callus lines carrying the empty vector and the rolC gene construct reached 106 and 146 mg/L, respectively. Moreover, the rolC gene construct promoted cell growth and overall cell productivity. The transgenic callus lines expressing the rolC gene exhibited higher levels of the following six isoflavonoids: daidzein, calycosin, formononetin, 4′-Ο-β-glucopyranosyldaidzin, maackiain and 6′-O-malonyl-3-O-β-D-glucopyranosylmaackiain. However, lower levels of genistin were observed in rolC calli than in those carrying the empty vector.     

Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration

To obtain insight into the mechanism of transferred DNA (T-DNA) integration in a long-lived tree system, we analysed 30 transgenic aspen lines. In total, 27 right T-DNA/plant junctions, 20 left T-DNA/plant junctions, and 10 target insertions from control plants were obtained. At the right end, the T-DNA was conserved up to the cleavage site in 18 transgenic lines (67%), and the right border repeat was deleted in nine junctions. Nucleotides from the left border repeat were present in 19 transgenic lines out of 20 cases analysed. However, only four (20%) of the left border ends were conserved to the processing end, indicating that the T-DNA left and right ends are treated mechanistically differently during the T-DNA integration process. Comparison of the genomic target sites prior to integration to the T-DNA revealed that the T-DNA inserted into the plant genome without any notable deletion of genomic sequence in three out of 10 transgenic lines analysed. However, deletions of DNA ranging in length from a few nucleotides to more than 500 bp were observed in other transgenic lines. Filler DNAs of up to 235 bp were observed on left and/or right junctions of six transgenic lines, which in most cases originated from the nearby host genomic sequence or from the T-DNA. Short sequence similarities between recombining strands near break points, in particular for the left T-DNA end, were observed in most of the lines analysed. These results confirm the well-accepted T-DNA integration model based on single-stranded annealing followed by ligation of the right border which is preserved by the VirD2 protein. However, a second category of T-DNA integration was also identified in nine transgenic lines, in which the right border of the T-DNA was partly truncated. Such integration events are described via a model for the repair of genomic double-strand breaks in somatic plant cells based on synthesis-dependent strand-annealing. This report in a long-lived tree system provides major insight into the mechanism of transgene integration.     

Analysis of T-DNA-<Emphasis Type="Italic">Xa21</Emphasis> loci and bacterial blight resistance effects of the transgene <Emphasis Type="Italic">Xa21</Emphasis> in transgenic rice

The genetic loci and phenotypic effects of the transgene Xa21, a bacterial blight (BB) resistance gene cloned from rice, were investigated in transgenic rice produced through an Agrobacterium-mediated transformation system. The flanking sequences of integrated T-DNAs were isolated from Xa21 transgenic rice lines using thermal asymmetric interlaced PCR. Based on the analysis of 24 T-DNA- Xa21 flanking sequences, T-DNA loci in rice could be classified into three types: the typical T-DNA integration with the definite left and right borders, the T-DNA integration linked with the adjacent vector backbone sequences and the T-DNA integration involved in a complicated recombination in the flanking sequences. The T-DNA integration in rice was similar to that in dicotyledonous genomes but was significantly different from the integration produced through direct DNA transformation approaches. All three types of integrated transgene Xa21 could be stably inherited and expressed the BB resistance through derived generations in their respective transgenic lines. The flanking sequences of the typical T-DNA integration consisted of actual rice genomic DNA and could be used as probes to locate the transgene on the rice genetic map. A total of 15 different rice T-DNA flanking sequences were identified. They displayed restriction fragment length polymorphisms (RFLPs) between two rice varieties, ZYQ8 and JX17, and were mapped on rice chromosomes 1, 3, 4, 5, 7, 9, 10, 11 and 12, respectively, by using a double haploid population derived from a cross between ZYQ8 and JX17. The blast search and homology comparison of the rice T-DNA flanking sequences with the rice chromosome-anchored sequence database confirmed the RFLP mapping results. On the basis of genetic mapping of the T-DNA- Xa21 loci, the BB resistance effects of the transgene Xa21 at different chromosome locations were investigated using homozygous transgenic lines with only one copy of the transgene. Among the transgenic lines, no obvious position effects of the transgene Xa21 were observed. In addition, the BB resistance levels of the Xa21 transgenic plants with different transgene copy numbers and on different genetic backgrounds were also investigated. It was observed that genetic background (or genome) effects were more obvious than dosage effects and position effects on the BB resistance level of the transgenic plants.     

Overexpression of Arabidopsis cytokinin oxidase/dehydrogenase genes AtCKX1 and AtCKX2 in transgenic Centaurium erythraea Rafn.

Cytokinin oxidase/dehydrogenase (CKX) is the only known enzyme involved in cytokinin catabolism. Genes coding for two Arabidopsis CKX isoforms, AtCKX1 and AtCKX2, were introduced separately into a binary cloning vector, immobilized into Agrobacterium tumefaciens strain GV3101, and introduced into root explants of centaury (Centaurium erythraea Rafn.). The integration of each transgene was confirmed by genomic PCR. Of the total transformed explants, 30 and 28.2 % of the transformants carried AtCKX1 and AtCKX2 transgenes, respectively. Of these transformants, 50 % exhibited expression of the AtCKX1 transgene, while 64 % of transformants exhibited expression of the AtCKX2 transgene. For all analysed AtCKX transgenic centaury lines, as well as for untransformed control plants, CKX activity was higher in roots than in shoots. Expression of AtCKX in most transgenic lines contributed to enhanced levels of CKX activity in root tissues; whereas, only a few lines demonstrated increased CKX activity in shoot tissues compared to those of control plants. Moreover, overexpression of AtCKX resulted in reduced morphogenetic potential in transgenic plants, but did not significantly affect biomass production in comparison to untransformed control plants.     

Production of insect-resistant transgenic rice plants for use in practical agriculture

Plant biotechnology provides a powerful solution to boost agricultural productivity and nutritional quality. The development process of a transgenic crop includes multiple steps that consist of gene isolation for a target trait, generation of T₀ transgenic crops, characterization of the transgene, evaluation of agronomic performance of transgenic crops, selection of elite transgenic lines and assessment of target trait efficacy. Here, we developed elite insect-resistant transgenic rice plants that may satisfy the standards of biosafety assessments. We made a construct with the insecticide cry1Ac gene for a target trait. A total of 310 T₀ transgenic lines were generated and underwent extensive analysis. We selected four T₃ lines that contain a single-copy transgene inserted into intergenic regions of the rice genome. During this process, we critically analyzed the transgenic lines with five checkpoints that include single copy of transgene, its integration into intergenic region, clean T-DNA arrangement, stability of transgene through generations and substantial equivalence of transgenic plants in agronomic traits other than insect resistance. Consequently, we obtained insect-resistant transgenic rice plants that can be used in practical agriculture.     

Expression of a rice chitinase gene in transgenic banana (��Gros Michel��, AAA genome group) confers resistance to black leaf streak disease

Transgenic banana (Musa acuminata ??Gros Michel??) integrating either of two rice chitinase genes was generated and its resistance to Black Leaf Streak disease caused by the fungus Mycosphaerella fijiensis was tested using a leaf disk bioassay. PCR screening indicated the presence of the hpt selectable marker gene in more than 90 % of the lines tested, whereas more than three quarters of the lines contained the linked rice chitinase gene resulting in a co-transformation frequency of at least 71.4 %. Further, a unique stable integration of the transgenes in each line revealed some false negative PCR results and the expected co-transformation frequency of 100 %. The transgene insert number per line ranged from 1 to 5 and single transgene insert lines (25 % of all) were identified. Considerable delay in disease development (up to 63 days post-incoculation) over a monitoring period of 108 days occurred in nine lines with extracellularly targeted chitinase out of 17 transgenic lines tested and their necrotic leaf area decreased by 73?C94 % compared to the untransformed susceptible control line. Finally, correlation between symptom development and rice chitinase expression was confirmed in two lines by Western analysis. The potential of rice chitinase genes to enhance resistance against M. fijiensis in banana was demonstrated as well as the usefulness of the leaf disk bioassay for early disease screening in transgenic banana lines.     

Integration, stability and expression of the E. coli phytase transgene in the Cassie line of Yorkshire Enviropig™

The genomic structure and generational stability of the transgene carried by the Cassie (CA) line of the transgenic Enviropig?, a prospective food animal, are reported here. This transgene is composed of the Escherichia coli phytase coding sequence regulated by the mouse parotid secretory protein promoter to direct secretion of phytase in the saliva. In the CA line the transgene integrated in chromosome 4 is present as a concatemer of three copies, two in a head to tail orientation and the third in a reverse orientation 3′ to the other copies with a 6 kbp deletion in the 5′ promoter region. The overall size of the integrated transgene complex is 46 kbp. During integration a 66 kbp segment of the chromosome was deleted, but a BLAST search of the segment from a GenBank clone did not reveal any essential genes. The transgene integration site was stable through 9 generations analyzed. Phytase activity in the saliva was similar among 11 day old hemizygous boars and gilts and remained relatively constant through nine generations of hemizygous pigs. However, as the pigs grew there generally was a gradual decrease in activity that stabilized when pigs reached the finisher phase of growth (4–6 months old). Homozygous pigs exhibited 1.5 fold higher phytase activity (P < 0.0001) than that of hemizygous littermates. Moreover, no differential salivary phytase activity was seen in hemizygotes arising from CA-Yorkshire and CA-Duroc breed outcrosses, suggesting that expression of the transgene is unaffected by genetic background. This data demonstrates that an exogenous phytase gene can be stably transmitted and expressed in the salivary glands of a domestic food animal.     

Overexpression of a Defensin Enhances Resistance to a Fruit-Specific Anthracnose Fungus in Pepper

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL⁻¹. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease.