Co-silencing of homologous transgenes in tobacco

Two transgenes inserted into different genomic positions can co-inactivate each other when they share homologous sequences while each of the two homologous transgenes is stably expressed in the absence of a second homologous copy. To evaluate the efficiency of such homology-dependent gene silencing (HDGS) effects, we have produced 19 tobacco transformants that contained a stably expressed NPTII transgene inserted into a single genomic locus, and have analysed the stability of each transgene in the presence of a second stably expressed homologous transgene. All transformants shared the coding region of the NPTII gene but individual transformants differed in transgene copy number, expression levels and in the continuity of the transgene homology due to the insertion of introns into the NPTII region as well as the use of different promoters and terminators for the design of the transgene constructs. We generated 189 progeny populations representing all possible dual combinations among the 19 lines and analysed the kanamycin resistance of 400 seedlings of each cross. Our data show (1) that gene silencing occurs at a relative low frequency when transgenic loci sharing an homology at the coding sequence level are combined, and (2) that neither the variation of this homology by insertion of introns in the coding sequence, or by changing the promoter and terminator of the construct, nor the variation in the expression level of the transgene, are decisive parameters modifying the efficiency of co-silencing between two NPTII transgenes.     

Assembly of an active chromatin structure during replication.

MSB cells were pulse labeled with 3H-thymidine and the isolated nuclei digested with either staphylococcal nuclease (to about 40% acid solubility) or DNase I (to 15% acid solubility). The purified, nuclease resistant single-copy DNA was then hybridized to nuclear RNA (nRNA). The results of these experiments show that actively transcribed genes are assembled into nucleosome-like structures within 5-10 nucleosomes of the replication fork and that they also acquire a conformation characteristic of actively transcribed nucleosomes (ie, a DNase I sensitive structure) within 20 nucleosomes of the fork. Assuming DNA sequence specific interactions are required for establishing a DNase I sensitive conformation on active genes during each round of replication, our results indicate that a specific recognition event can occur very rapidly and very specifically in eukaryotic cells. The results are discussed in terms of the possible mechanisms responsible for propagating active, chromosomal conformations from mother cells to daughter cells.     

Inheritance and effectiveness of two transgenes determining PVY resistance in progeny from crossing independently transformed tobacco lines

Genetic transformation of plants allows us to obtain improved genotypes enriched with the desired traits. However, if transgenic lines were to be used in breeding programs the stability of inserted transgenes is essential. In the present study, we followed the inheritance of transgenes in hybrids originated from crossing two transgenic tobacco lines resistant to Potato virus Y (PVY): MN 944 LMV with the transgene containing Lettuce mosaic virus coat protein gene (LMV CP) and AC Gayed ROKY2 with PVY replicase gene (ROKY2). Progeny populations generated by successive self-pollination were analyzed with respect to the transgene segregation ratio and resistance to Potato virus Y in tests carried out under greenhouse conditions. The presence of the virus in inoculated plants was detected by DAS-ELISA method. The results demonstrated the Mendelian fashion of inheritance of transgenes which were segregated independently and stably. As a result, we obtained T₄ generation of hybrid with both transgenes stacked and which was highly resistant to PVY.     

SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性,以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台,本研究将人工优化合成的SARS-CoV突刺蛋白(S蛋白)受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明,外源融合基因已整合到烟草叶绿体基因组中,并获得同质化。Western杂交分析表明,重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达,且主要以可溶性单体形式存在。ELISA分析表明,在不同生长阶段、不同生长部位和不同时间点烟草叶片中,重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2%TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达,有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。     

Trans-activation of an artificial dTam3 transposable element in transgenic tobacco plants

In Antirrhinum majus only autonomous Tam3 transposons have been characterized. We investigated whether an artificial dTam3 element, with a deletion in the presumptive transposase coding region, can be trans-activated in tobacco by an activator Tam3 element, which was immobilized by the deletion of one inverted repeat. A phenotypic assay based on restored hygromycin resistance demonstrates that a dTam3 element harbouring a bacterial plasmid can be trans-activated with a low frequency. Molecular analysis confirms that the dTam3 element has been excised from the HPTII marker gene. Reintegration of the dTam3 element into the tobacco genome is detected only in one out of six hygromycin-resistant plants analysed. PCR analysis of empty donor sites shows that excision of the dTam3 element in tobacco results in rearrangements (deletions and additions), that have been shown to be characteristic of Tam3 excision in the original host Antirrhinum majus. This trans-activation assay allowed us to establish that, in contrast to what has been detected in Antirrhinum majus, a periodical temperature shift down to 15°C does not enhance dTam3 transposition in regenerating tobacco calli.     

Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F₂ plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F₃ and F₄ generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.     

Nature-inspired engineering of an artificial ligase enzyme by domain fusion

The function of most proteins is accomplished through the interplay of two or more protein domains and fine-tuned by natural evolution. In contrast, artificial enzymes have often been engineered from a single domain scaffold and frequently have lower catalytic activity than natural enzymes. We previously generated an artificial enzyme that catalyzed an RNA ligation by >2 million-fold but was likely limited in its activity by low substrate affinity. Inspired by nature''s concept of domain fusion, we fused the artificial enzyme to a series of protein domains known to bind nucleic acids with the goal of improving its catalytic activity. The effect of the fused domains on catalytic activity varied greatly, yielding severalfold increases but also reductions caused by domains that previously enhanced nucleic acid binding in other protein engineering projects. The combination of the two better performing binding domains improved the activity of the parental ligase by more than an order of magnitude. These results demonstrate for the first time that nature''s successful evolutionary mechanism of domain fusion can also improve an unevolved primordial-like protein whose structure and function had just been created in the test tube. The generation of multi-domain proteins might therefore be an ancient evolutionary process.     

The expression of tomato prosystemin gene in tobacco plants highly affects host proteomic repertoire

Systemin, an octadecapeptide isolated from tomato, is a primary signal molecule involved in the local and systemic responses to pest attack, elicited by activation of a set of defence genes. It derives from processing of prosystemin, a prohormone of almost 200 amino acids. Prosystemin orthologues have been found in other Solanaceae species but not in tobacco, where are present hydroxyproline-rich peptides functionally but not structurally related to tomato systemin. Molecular events leading to the release of signalling peptides from protein precursors are unknown in plants; the occurrence of a family of signal molecules suggests that initiation of wound response may involve different processing mechanisms. It has been previously shown that the protein product from an engineered tomato prosystemin gene is processed in tobacco, thus suggesting that the components responsible for its post-translational modifications are present in this species. By analyzing analysing the proteome repertoire of transformed tobacco plant leaves with 2-DE, here we demonstrate that the constitutive expression of the tomato prosystemin gene highly affected host protein synthesis. In particular, engineered plants showed a number of differentially synthesized proteins that were identified by PMF MALDI-TOF and microLC-ESI-IT-MS/MS experiments as polypeptide species involved in protection from pathogens and oxidative stress, or in carbon/energy metabolism. Significant differences in over-produced proteins were observed with respect to previous data reported on systemin-engineered tomato plants. Our results strongly support the need of using proteomic approaches during systematic analysis of plant tissues to investigate the principle of substantial equivalence in transgenic plants expressing a transgene coding for a signalling molecule.     

Efficient expression of transgenes in adult zebrafish by electroporation

Background  

Expression of transgenes in muscle by injection of naked DNA is widely practiced. Application of electrical pulses at the site of injection was demonstrated to improve transgene expression in muscle tissue. Zebrafish is a precious model to investigate developmental biology in vertebrates. In this study we investigated the effect of electroporation on expression of transgenes in 3–6 month old adult zebrafish.