Integration Of Heterologous Genes Research Articles

Increasing of barley productivity and adaptability by using genetic modification technologies

The review presents the achievements in the research of barley stress tolerance molecular mechanisms to improve its economically valuable traits as a crop of agricultural production. Possible genetic engineering approaches developed in order to increase the barley resistance to abiotic stresses have been described. Special attention is paid to the genome improvement through the integration of heterologous genes. The targeted genes and promotors perspective for transformations, their efficiency in dependence to other transformation factors have been summarized and analyzed. The latest technologies of targeted mutagenesis used for genome editing (ZFN, TALEN and CRISPR/Cas9 systems) are observed. The examples of creation of new barley forms with increased resistance to drought, soil acidity, aluminum toxicity and salt stress by using gene technologies are given. It is shown that genetic modification allows not only to accelerate the experiment process of new genotypes creation, but also represents a research tool for the analysis and identification of gene functions. А large number of sequences encoding valuable traits of barley were identified by means of silencing and RNA interference techniques. The prospects of development of post-genomic technologies for use in practical breeding of this culture are discussed.

Conjugational delivery of chromosomal integrative constructs for gene expression in the carbendazim-degrading Rhodococcus erythropolis D-1

Rhodococcus strains not only have been widely used in industries but also have a potential ability of producing new structural natural products. Integration of heterologous genes into chromosomes of Rhodococcus strains for gene expression can facilitate the studies and applications of these strains. A conjugation system was optimized in order to transfer enhanced green fluorescent protein (EGFP) encoding gene as a reporter from Escherichia coli into Rhodococcus erythropolis D-1. The influence of three native ribosome binding sites (RBSs) and two designed RBSs on the target protein production in R. erythropolis D-1 was also characterized. An efficient conjugation system of R. erythropolis D-1 was established to integrate EGFP gene into its chromosome. Among of five RBSs, RBS3 showed the highest translational activity in R. erythropolis D-1.

Integration of heterologous genes in several yeast species using vectors containing a Hansenula polymorpha-derived rDNA-targeting element.

A method that has been successfully used to generate recombinant Hansenula polymorpha strains by transformation with rDNA-targeting vectors was applied in the present study to a range of alternative yeast hosts, using vectors with an H. polymorpha-derived integration sequence. The dimorphic yeast Arxula adeninivorans, which is currently being assessed for heterologous gene expression, was the main focus of the study. As in H. polymorpha, it was possible to co-integrate more than a single plasmid carrying an expressible gene. Additionally, the vectors were examined in two further species, Pichia stipitis and Saccharomyces cerevisiae. Based on these results the design of a 'universal' fungal vector appears to be feasible.

Location effects of a reporter gene on expression levels and on native protein synthesis in Lactococcus lactis and Saccharomyces cerevisiae.

The engineering of industrially important genetically modified organisms by the integration of heterologous genes into the chromosome is often the method of choice for several reasons concerned with long-term stability, homogeneous population distribution, and the enabling of selection without the addition of antibiotics. However, integration may disrupt endogenous gene expression, giving rise to increased levels of toxic metabolic byproducts or activating otherwise silent genes. The position of integration of a foreign gene in the chromosome can also influence its expression levels, and this effect will be of relevance in terms of optimizing protein production parameters. In this study, we determine how the random integration of a foreign reporter gene might affect expression levels and assess the use of proteome analysis to investigate possible effects on synthesis of endogenous proteins in two important food-relevant microorganisms, Saccharomyces cerevisiae and Lactococcus lactis. Eleven L. lactis integrants carrying the gusA gene were analyzed, and expression levels were found to vary by a factor of threefold in contrast to expression levels of lacZ in 18 S. cerevisiae integrants, which showed a 14-fold variation. Of relevance to industry is whether any changes in expression levels might occur as a consequence of storage of the modified strains. Here it is also shown that the above differences in expression levels were not significantly affected by storage of frozen cultures over a period of several months. Analysis of the protein composition of the yeast and lactococcal integrant strains by separation on one-dimensional (1D) and 2D gels showed no significant variations in position beyond those observed in control samples.