Removal of the Antibiotic Resistance Genes in Chloroplast Transformation

Introduction

Currently, antibiotic genes play an important role in chloroplast genetic engineering as selectable marker genes. But the transfer of antibiotic resistance genes into microbes has the potential to create "superbugs" that are resistant to clinically used antibiotics. In addition, the high expression of antibiotic resistance gene protein products in homogenized plants may impose a burden on the plant's own metabolism. Although the inheritance of chloroplasts in most crops follows the laws of maternal inheritance, the possibility of transfer of selectable marker genes to wild-type plants or microorganisms cannot be completely ruled out. Scientists have already achieved gene excision of antibiotic markers in tobacco plants. Therefore, as with nuclear transformation, removal of antibiotic resistance genes from the transformed chloroplast genome is clearly the easiest and most feasible approach.

Fig. 1. The cotransformation-segregation method to remove selectable marker genes from transplastomic plants. (Chong-Pérez B, et al., 2013)

Our Services

Compared with natural plant variability or changes related to environmental and agronomic factors, the insertion of transgenes has very little effect on the plant transcriptome, proteome or metabolome. And the same is true in chloroplast genetic transformation. At the same time, there are few effective selection markers that can be used for chloroplast transformation. With the support of the chloroplast genetic engineering platform, Lifeasible is committed to providing customers with high-quality antibiotic resistance gene removal services to achieve the removal of antibiotic resistance genes or reporter genes from transgenic plants after chloroplast transformation, addressing regulatory issues and public concerns. Our engineers have developed the following mature strategies for removing selectable marker genes, and our aim is to be your most professional and reliable partner.

· Homology-Based Excision by Direct Repeats
Any sequence between forward repeats on the chloroplast genome can be excised by homologous recombination, depending on the presence of directly repeated identical plastid DNA sequences. We have successfully applied this strategy to the deletion of the aadA gene in the chloroplast genomes of C. reinhardtii and tobacco transgenic plants.

· Excision by Phage Site-Specific Recombinase
Here, we implement this strategy through a two-step protocol. The first is to produce transplastid plants with a selectable marker gene flanked by two directly directed recombinase target sites. Thereafter, marker-removed plants are obtained when recombinase activity is introduced by nuclear transformation of a gene encoding a plastid-targeted recombinase.

· Transient Co-Integration of Marker Genes
We used a knockout mutant to aid in visual complementation analysis to remove antibiotic resistance genes based on the instability of the marker gene and the gene of interest co-integrated into the plastid genome by double crossover of the targeted sequence.

· Common Conversion-Separation Method
We insert two different transformation vectors (one vector carries the selectable marker gene, the other vector carries the target gene) into the chloroplast genome for co-transformation, and then isolates to obtain marker-free transplastid plants.

Advantages of Removing the Antibiotic Resistance Gene

• A variety of efficient, safe and effective strategies are available.
• Fewer technical and biosafety constraints than nuclear transformation.
• Any possible positional or pleiotropic effects can be prevented from causing unexpected changes.
• Can facilitate advanced studies requiring isolation of double mutants in distant plastid genes and replacement of missing loci with site-directed mutagenic alleles.
• Marker-free plants can be generated directly in the first generation without the need for further transformation or crossing.
• The excision of the selectable marker gene does not affect the integrity of other functional genes in the chloroplast.

Lifeasible has successfully employed multiple strategies for chloroplast gene transformation without using antibiotic resistance genes. With our team's extensive experience in antibiotic-free chloroplast transformation, we are able to provide services for the removal of antibiotic resistance genes from transplastid plants to our global clients. If you are interested in our services, please do not hesitate to contact us for more information.

References

1. Chong-Pérez B, Angenon G. (2013) Strategies for generating marker-free transgenic plants[J]. Genetic Engineering. 2: 17-48.
2. Iamtham S, Day A. (2000) Removal of antibiotic resistance genes from transgenic tobacco plastids[J]. Nature biotechnology. 18(11): 1172-1176.