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     

外源基因在转基因动物中遗传和表达的稳定性

转基因技术经过近半个世纪的发展,已成为当今生物技术研究的热点。近10多年来,与核移植技术的结合,转基因效率大大提高,携带有不同外源基因的不同种类的转基因动物迅速增加。但是,成功获得转基因动物并不是转基因动物研究的最终目的,如何利用转基因技术为人类的需求服务才是科研人员始终面对的课题。在畜牧生产领域,通过转基因技术培育家畜新品种是转基因技术应用的重要体现,在我国这方面已经引起了广泛关注。但迄今为止,外源基因在转基因动物中遗传和表达的稳定性仍然是亟待解决的问题,究其原因,这主要与位置效应、外源基因的表观遗传学修饰和遗传效率相关,文章结合目前的研究进展和本实验室的研究结果,从这3方面阐述其作用机制,期望为转基因动物遗传育种向产业化的迈进提供一定的理论探讨。     

Axenic mycorrhization of wild type and transgenic hybrid aspen expressing T-DNA indoleacetic acid-biosynthetic genes

 We describe and document the in vitro synthesis of ectomycorrhiza between roots of wild type and transgenic aspen (Populus tremula × P. tremuloides), expressing Agrobacterium tumefaciens T-DNA indoleacetic acid (IAA)-biosynthetic genes, and Amanita muscaria. Plantlets were raised from tissue culture. The root system of approximately 4-week-old plantlets was transferred to Petri dishes and incubated together with fungal mycelia under sterile conditions. Ectomycorrhiza showing both a well developed hyphal mantle and Hartig net were established within 3 to 4 weeks. Formation and morphology of ectomycorrhiza were not affected by the transformation of aspen, expressing the IAA biosynthetic genes in roots. As both hybrid aspen and fungal cells can be genetically engineered, this system offers a new approach to the study of mycorrhizal symbioses. Received: 19 January 1996 / Accepted: 23 January 1996     

Matrix attachment region elements have small and variable effects on transgene expression and stability in field‐grown Populus

Matrix attachment regions (MARs) are thought to buffer transgenes from the influence of surrounding chromosomal sequences, and therefore to reduce transgene silencing and variation in expression. The statistical properties of more than 400 independent transgenic events produced in Populus, with and without flanking MAR elements from the tobacco root gene RB7, were analysed. The expression of two reporter genes in two poplar clones during three phases of vegetative growth, and the association of T‐DNA characteristics with expression, was examined. It was found that MARs did not show a consistent effect on transgene expression levels; they had no effect on the green fluorescent protein (GFP) reporter gene, but reduced expression in the Basta resistance (BAR) reporter gene by 23%. The presence of MARs reduced expression variability within transformant populations, apparently by reducing the number of silenced or weakly expressing events. Transgene expression was highly stable over vegetative growth cycles that spanned 3 years of growth in the glasshouse and field, but MARs showed no association with the strength of correlations in expression over the years. Nonetheless, MARs increased the correlation in expression between a p35S::GFP and prbcS::BAR transgene linked on the same vector, but the effect was small and varied between the years. The presence of MARs had no effect on the transgene copy number, but was positively associated with T‐DNA truncations, as well as with the formation of direct over inverted repeats at the same chromosomal locus.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

rol-Gene expression in transgenic aspen (Populus tremula) plants results in accelerated growth and improved stem production index

Stem and trunk growth, axillary bud break and branching habits are extremely important parameters of wood production in forest trees. The possibility of altering tree form by transformation with genes responsible for hormone biosynthesis and/or activity is most attractive. We examined four different phenotypically selected transgenic clones of a model tree –Populus tremula– expressing rol genes from Agrobacterium rhizogenes under their native promoters. Several of the observed phenotypic modifications were correlated with rol-gene expression, including breaking of stem apical dominance which resulted in the development and branching of up to four axillary buds per explant, as compared to a lack of axillary bud break in a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. rol-Transgenic plants also exhibited a higher cumulative stem length and enhanced growth rate, and hence a higher stem production index. During their first and second years in the greenhouse, rol-transgenic aspen plants exhibited enhanced growth and delayed winter dormancy relative to non-transformed plants. Although initially rol-transgenic plants had smaller, wrinkled leaves, these changes were not observed in the 2-year-old plants, which exhibited a phenotypically true-to-type leaf shape. Received: 13 September 1998 / Accepted: 15 April 1999     

Expression of Vitreoscilla haemoglobin in hybrid aspen (Populus tremula x tremuloides)

We describe the first ever expression of Vitreoscilla haemoglobin (VHb) in an economically important boreal woody plant hybrid aspen (Populus tremula x tremuloides). VHb has mainly been expressed in biotechnologically important unicellular organisms of both prokaryotic and eukaryotic origin. VHb expression, in this study, was analysed under different greenhouse cultivation conditions and under elevated UV-B illumination. Microscope analyses of leaves grown under optimized conditions revealed significant differences both in cell structure and size when the transgenic VHb lines were compared with the control lines. VHb lines displayed a higher relative volume of mitochondria and a significantly enhanced accumulation of starch in chloroplasts, all of which pointed towards changes in cellular energy production. Under elevated UV-B illumination, the differences between VHb lines became evident. Some specific VHb lines had elevated levels of total flavonoids, individual quercetin, kaempferol- and myricetin-derivatives relative to controls and other transgenic lines. This observation may reflect the availability of extra energy resources for secondary metabolite production and possibly an enhanced protection ability of these transgenic lines against UV-B illumination. Thus, all these findings point to changes in the energy metabolism of VHb lines. In the cultivation conditions tested this observation did not, however, result in a general improvement of elongation growth.     

Agrobacterium-mediated transformation of quaking aspen (Populus tremuloides) and regeneration of transgenic plants

Summary Agrobacterium-mediated gene transformation of Populus tremuloides Michx was accomplished by co-cultivation of leaf disks excised from greenhouse plants with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase (NPT II) and ß-glucuronidase (GUS) genes. Shoot regeneration in the presence of kanamycin was achieved when thidiazuron (TDZ) was used as a plant growth regulator. Transformation was verified by amplification of NPT II and GUS gene fragments from genomic DNA of transgenic plants with polymerase chain reaction (PCR) and integration of these genes into nuclear genome of transgenic plants was confirmed by genomic Southern hybridization analysis. Histochemical assay revealed the expression of GUS gene in leaf, stem and root tissues of transgenic plants, further confirming the integration and expression of T-DNA in these plants. This protocol allows effective transformation and regeneration of quaking aspen using greenhouse-grown materials as an explant source. Whole plant regeneration from cuttings of fieldgrown mature quaking aspen and hybrid poplar (P. alba x P. grandidentata) was also readily achieved by using this protocol, which represents a potential system for producing transgenic quaking aspen and hybrid poplar of valuable genotypes.Abbreviations AMV RNA4 Alfalfa mosaic virus RNA4 - BA 6-benzyladenine - CaMV cauliflower mosaic virus - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediaminetetraacetic acid - FAA formalin-acetic acid-alcohol - GUS ß-glucuronidase - NAA 1-naphthylacetic acid - NPT II neomycin phosphotransferase II - PCR polymerase chain reaction - SDS sodium dodecyl sulphate - TE Tris-Cl/EDTA - TDZ N-phenyl-N-1,2,3-thiadiazol-5-yl-urea (thidiazuron) - WPM woody plant medium (Lloyd and McCown 1980) - X-GLUC 5-bromo-4-chloro-3-indolyl-ß-glucuronic acid     

Genetic linkage mapping in aspen (Populus tremula L. and Populus tremuloides Michx.)

A large number of simple sequence repeat (SSR) marker-containing genetic maps are available for several Populus species. For aspen however, no SSR-containing map has been published so far. In this study, genetic linkage mapping was carried out with an interspecific mapping pedigree of 61 full-sib hybrids of European × quaking aspen (Populus tremula L. × Populus tremuloides Michx.), using the two-way pseudo-testcross strategy. Amplified fragment-length polymorphism (AFLP) and SSR markers were used for mapping, resulting in the first SSR-containing genetic linkage maps for aspen. The maps allow comparisons with a Populus consensus map and other published genetic maps of the genus Populus. The maps showed good collinearity to each other and to the Populus consensus map and provide a direct link to the Populus trichocarpa genomic sequence. Sex as a morphological trait was assessed in the mapping population and mapped on a non-terminal position of linkage group XIX on the male P. tremuloides map.     

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.     

Molecular characterization of of alpha-gliadin genes from wild emmer wheat (Triticum dicoccoides).

According to the two distal and conserved regions of known alpha-gliadin genes, gene-specific primers for alpha-gliadin were designed, and the coding regions of four gliadin genes (i.e. GliTd-1, GliTd-2, GliTd-3 and GliTd-4) with the length of about 800 bp were isolated from the genomic DNA of wild emmer wheat (Triticum dicoccoides). No introns were observed. Sequence comparison indicated that these genes should be classified as alpha-gliadins. GliTd-3 (GenBank accession No.DQ140351) and GliTd-4 (DQ140352) were potentially functional, whereas GliTd-1 (DQ140349) and GliTd-2 (DQ140350) were both pseudogenes by the definition of in-frame stop codons and frameshifts. Six conserved cysteine residues were observed. Sequence analysis suggested that the motif units of repetitive domain for the four newly detected genes were different from the known genes, and the QQQP sequence before the position 60 was more toxic to coeliac patients. Codons for proline were strongly biased. Codons (CAG and CAA) for glutamine were clustered into the specific regions, and the high percentage of pseudogenes resulted from the mutation of CAG --> TAG.     

Extrafloral nectaries in aspen (Populus tremuloides): heritable genetic variation and herbivore-induced expression

BACKGROUND AND AIMS: A wide variety of plants produce extrafloral nectaries (EFNs) that are visited by predatory arthropods. But very few studies have investigated the relationship between plant genetic variation and EFNs. The presence of foliar EFNs is highly variable among different aspen (Populus tremuloides) genotypes and the EFNs are visited by parasitic wasps and predatory flies. The aim here was to determine the heritability of EFNs among aspen genotypes and age classes, possible trade-offs between direct and indirect defences, EFN induction following herbivory, and the relationship between EFNs and predatory insects. METHODS: EFN density was quantified among aspen genotypes in Wisconsin on trees of different ages and broad-sense heritability from common garden trees was calculated. EFNs were also quantified in natural aspen stands in Utah. From the common garden trees foliar defensive chemical levels were quantified to evaluate their relationship with EFN density. A defoliation experiment was performed to determine if EFNs can be induced in response to herbivory. Finally, predatory arthropod abundance among aspen trees was quantified to determine the relationship between arthropod abundance and EFNs. KEY RESULTS: Broad-sense heritability for expression (0.74-0.82) and induction (0.85) of EFNs was high. One-year-old trees had 20% greater EFN density than 4-year-old trees and more than 50% greater EFN density than > or =10-year-old trees. No trade-offs were found between foliar chemical concentrations and EFN density. Predatory fly abundance varied among aspen genotypes, but predatory arthropod abundance and average EFN density were not related. CONCLUSIONS: Aspen extrafloral nectaries are strongly genetically determined and have the potential to respond rapidly to evolutionary forces. The pattern of EFN expression among different age classes of trees appears to follow predictions of optimal defence theory. The relationship between EFNs and predators likely varies in relation to multiple temporal and environmental factors.     

Molecular analysis of herbivore-induced condensed tannin synthesis: cloning and expression of dihydroflavonol reductase from trembling aspen (Populus tremuloides)

In order to study condensed tannin synthesis and its induction by herbivory, a dihydroflavonol reductase (DFR) cDNA was isolated from trembling aspen (Populus tremuloides). Bacterial overexpression demonstrated that this cDNA encodes a functional DFR enzyme, and Southern analysis revealed that DFR likely is a single-copy gene in the aspen genome. Aspen plants that were mechanically wounded showed a dramatic increase in DFR expression after 24 h in both wounded leaves and unwounded leaves on wounded trees. Feeding by forest tent caterpillar (Malacosoma disstria) and satin moth (Leucoma salicis) larvae, and treatment with methyl jasmonate, all strongly induced DFR expression. DFR enzyme activity was also induced in wounded aspen leaves, and phytochemical assays revealed that condensed tannin concentrations significantly increased in wounded and systemic leaves. The expression of other genes involved in the phenylpropanoid pathway were also induced by wounding. Our findings suggest that the induction of condensed tannins, compounds known to be important for defense against herbivores, is mediated by increased expression of DFR and other phenylpropanoid genes.     

Matrix attachment regions increase transgene expression levels and stability in transgenic rice plants and their progeny

To investigate the effect of matrix attachment regions (MARs) on transgene expression levels and stability in cereal crops, we generated 83 independent transgenic rice callus lines containing a gusA expression cassette either as a simple expression unit, or flanked with MARs from tobacco (Rb7) or yeast (ARS1). Transgenic rice plants were regenerated from these callus lines and analysed at the structural and expression levels over two generations. In the first generation (T₀), both Rb7 and ARS1 MARs significantly increased transgene expression levels. In the populations of plants containing MARs, we observed a significant reduction in the number of non-expressing lines compared to the population of plants without MARs. However, variation in β-glucuronidase (GUS) expression levels between independent lines was similar both in the presence and absence of flanking MARs. In the presence of MARs, GUS activity increased in proportion to transgene copy number up to 20 copies, but was generally reduced in lines carrying a higher copy number. In the population of plants without MARs, there was no correlation between expression level and transgene copy number. In the second generation (T₁), transgene expression levels were significantly correlated with those of the T₀ parents. The Rb7 MARs significantly improved the stability of transgene expression levels over two generations, and therefore appear to offer protection against transgene silencing. Our study shows that the exploitation of MARs may be an important strategy for stabilising transgene expression levels in genetically engineered cereals.     

Genomic stability and long-term transgene expression in poplar

Stable expression of foreign genes over the entire life span of a plant is important for long-lived organisms such as trees. For transgenic forest trees, very little information is available on long-term transgene expression and genomic stability. Independent transgenic lines obtained directly after transformation are initially screened in respect to T-DNA integration and transgene expression. However, very little consideration has been given to long-term transgene stability in long-lived forest trees. We have investigated possible genome wide changes following T-DNA integration as well as long-term stability of transgene expression in different transgenic lines of hybrid aspen (Populus tremula × Populus tremuloides) that are up to 19 years old. For studies on possible genome wide changes following T-DNA integration, four different independent rolC-transgenic lines were subjected to an extensive AFLP study and compared to the non-transgenic control line. Only minor genomic changes following T-DNA integration could be detected. To study long-term transgene expression, six different independent rolC-transgenic lines produced in 1993 and since that time have been kept continuously under in vitro conditions. In addition, 18 transgenic plants belonging to eight independent rolC-transgenic lines transferred to glasshouse between 1994 and 2004 were chosen to determine the presence and expression of the rolC gene. In all transgenic lines examined, the rolC gene could successfully be amplified by PCR tests. Both, the 19 years old tissue cultures and the up to 18 years old glasshouse-grown trees revealed expression of the rolC transgene, as demonstrated by the rolC-phenotype and/or northern blot experiments confirming long-term transgene expression.