Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Abstract

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACpl8, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organellespecific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology

“…[10]), enabled plant chloroplasts to be transformed without the use of isolated plastids. Chloroplast genetic engineering was accomplished through autonomously replicating chloroplast vectors in dicot plastids [11] and transient expression in monocot plastids [12], and by stable integration of selectable marker genes into the chloroplast genomes of Chlamydomonas reinhardtii and tobacco using the gene gun [13,14]. However, it is only recently that genes conferring agronomically valuable traits have been introduced via chloroplast genetic engineering.…”

“…Selectable markers and reporters aadA Aminoglycoside-3′-adenylyltransferase [14] nptII Neomycin phosphotransferase [52] codA Cytosine deaminase [53] BADH Betaine aldehyde dehydrogenase [4] uidA β-glucoronidase [12] cat Chloramphenicol acetyl transferase [9,11] gfp Green fluorescent protein [24,54] aadA:gfp Selectable or screenable fusion protein [47] Plant traits: herbicide resistance aroA Glyphosate resistance [2,19] bar Bialaphos resistance [18,20] Insect resistance…”

Containment of herbicide resistance through genetic engineering of the chloroplast genome

“…The discovery of the gene gun as a transformation device opened the possibility of direct plastid transformation 12 . Transient expression of foreign genes in plastids of dicots 13,14 and monocots 15 , prolonged foreign gene expression using autonomously replicating chloroplast expression vectors 13 , and stable integration of a selectable marker into the tobacco chloroplast genome 16 were accomplished using the gene gun. Tobacco plants resistant to certain insects were obtained by integrating the cryIAc gene into the tobacco chloroplast genome 17 .…”

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

“…The translation of native polycistrons without the need for processing to monocistrons has been demonstrated (Barkan, 1988;Zoschke and Barkan, 2015), but the similarity of this process using heterologous polycistrons engineered via the chloroplast genome offered even more direct evidence for this process (De Cosa et al, 2001;Quesada-Vargas et al, 2005). The insertion of replication origins into chloroplast vectors offered further insight into minimal sequences required to study this process (Daniell et al, 1990). Therefore, in this study, we use transgenes, chloroplast genome sequences, and cutting-edge tools to understand the process of translation in chloroplasts.…”