Development of chloroplast transformation and gene expression regulation technology in land plants

Abstract

Chloroplasts in land plants have their own small circular DNA that is presumed to have originated from cyanobacteria-related endosymbionts, and the chloroplast genome is an attractive target to improve photosynthetic ability and crop yield. However, to date, most transgenic or genetic engineering technologies for plants are restricted to manipulations of the nuclear genome. In this review, we provide a comprehensive overview of chloroplast genetic engineering and regulation of gene expression from the perspective of history and biology, focusing on current and latest methods. In addition, we suggest techniques that may regulate the chloroplast gene expression at the transcriptional or post-transcriptional level.

Keywords: CRISPR/Cas; aptamer; chloroplast genome; dPPR; gene expression; transformation.

Figure 1

Summary of the chloroplast transformation technologies and potential regulation methods for chloroplast gene expression. (A) Technologies that introduce target genes into chloroplasts include biolistics and nanoparticle-mediated transformation. Biolistics and nanoparticle-mediated methods require that foreign plasmid DNA be adhered to the surface of metal particles or wrapped in nanoparticles and then DNA be mediated by power of free-power into chloroplasts or nucleus. PEG can change the structure of cell membranes such that foreign plasmid DNA can enter plant cells and nucleus. The Agrobacterium infection method transforms the recombinant plasmid into competent cells of Agrobacterium and then infect plant cells with Agrobacterium to achieve nuclear transformation. (B) I: Regulation of chloroplast gene expression at the DNA level. Target genes can directly enter the chloroplast and regulate gene expression at the DNA level through CRISPR/Cas9 techniques via the HR method or foreign DNA functions as an independent minichromosome. II: Regulation of chloroplast gene expression at the RNA level. After the dPPR gene enters the recipient cell, it replicates and expresses dPPR protein. The fused transit peptide of the dPPR protein guides it to enter the chloroplast through the TOC/TIC transport complex. TOC, translocase at the outer envelope membrane of the chloroplast; TIC, translocase at the inter-chloroplast membrane. The dPPR protein directly binds with mRNA to regulate the expression of chloroplast genes. III: Regulation of chloroplast gene expression. After the target gene enters the recipient cell, it replicates and expresses in the nucleus to construct the aptamer. The aptamer with fused transit peptide can be guided into the chloroplast and interacts with the corresponding chloroplast protein to affect the chloroplast protein function.