Chloroplast Vector Systems for Biotechnology Applications

Abstract

Chloroplasts are ideal hosts for expression of transgenes. Transgene integration into the chloroplast genome occurs via homologous recombination of flanking sequences used in chloroplast vectors. Identification of spacer regions to integrate transgenes and endogenous regulatory sequences that support optimal expression is the first step in construction of chloroplast vectors. Thirty-five sequenced crop chloroplast genomes provide this essential information. Various steps involved in the design and construction of chloroplast vectors, DNA delivery, and multiple rounds of selection are described. Several crop species have stably integrated transgenes conferring agronomic traits, including herbicide, insect, and disease resistance, drought and salt tolerance, and phytoremediation. Chloroplast-derived vaccine antigens against bacterial (cholera, tetanus, anthrax, plague, and Lyme disease), viral (canine parvovirus [CPV] and rotavirus), and protozoan (amoeba) pathogens have been evaluated by immune responses, neutralizing antibodies, and pathogen or toxin challenge in animals. Chloroplasts have been used as bioreactors for production of biopolymers, amino acids, and industrial enzymes. Oral delivery of plant cells expressing proinsulin (Pins) in chloroplasts offered protection against development of insulitis in diabetic mice; such delivery eliminates expensive fermentation, purification, low temperature storage, and transportation. Chloroplast vector systems used in these biotechnology applications are described.

Complete plastid genome sequence of the chickpea (Cicer arietinum) and the phylogenetic distribution of rps12 and clpP intron losses among legumes (Leguminosae)

“…Plastid transgenic lines also lack gene silencing (DeCosa et al, 2001;Lee et al, 2003), position effect due to site specific transgene integration and pleiotropic effects due to subcellular compartmentalization of transgene products (Lee et al, 2003;Daniell et al, 2001;Leelavathi et al, 2003); concerns of transgene silencing, position effect and pleiotropic effects are often encountered in nuclear genetic engineering. Therefore, transgenes have been integrated into plastid genomes to confer valuable agronomic traits, including herbicide resistance (Daniell et al, 1998), insect resistance (McBride et al, 1995;DeCosa et al, 2001), disease resistance (DeGray et al, 2001), drought tolerance (Lee et al, 2003), salt tolerance (Kumar et al, 2004), phytoremediation (Ruiz et al, 2003;Hussein et al, 2007) or expression of various therapeutic proteins or biomaterials (Verma and Daniell, 2007;Kamarajugadda and Daniell, 2006;Daniell et al, 2005). However, soybean is the only legume that has been transformed via the plastid genome so far (Dufoumantel et al, 2004(Dufoumantel et al, , 2005 and more genome sequence information is needed to facilitate plastid genetic engineering in other economically important legumes.…”

High Levels of Bioplastic Are Produced in Fertile Transplastomic Tobacco Plants Engineered with a Synthetic Operon for the Production of Polyhydroxybutyrate

“…Gene abbreviations can be found in Table I Staub and Maliga, 1994a;Eibl et al, 1999). However, researchers still routinely used regulatory elements including promoters and untranslated regions with homology to sequences within the host plastome (Verma and Daniell, 2007), often because these were the only sequences readily available for use. While our experiments were in progress, the magnitude of the recombination issue was realized by several laboratories (Huang et al, 2002;Rogalski et al, 2006;Zhou et al, 2008;Gray et al, 2009), including our own.…”

Transplastomic plants for innovations in agriculture. A review

“…Although several selection markers and protocols have been developed, those based on aadA (encoding aminoglycoside 3″-adenylyltransferase, EC 2.7.7.47, and conferring spectinomycin and streptomycin resistance to bacteria by detoxification) are the most frequent ones (Table 1), most likely because they require low expression levels to confer phenotypic resistance. Detailed description and historical overview of the used vectors including promoters, effective selection markers, reporter genes, their insertion, and eventual removal are provided by recent reviews (Maliga 2003(Maliga , 2004Koop et al 2007;Verma and Daniell 2007;Ruhlman et al 2010;Day and Goldschmidt-Clermont 2011;Maliga and Bock 2011;Ahmad and Mukhtar 2013;Hanson et al 2013;Vafaee et al 2014).…”

“…These include (1) absence of gene silencing, epigenetic, and/or position effects, which eliminates the high variation in gene expression and thus in protein accumulation levels among independent transgenic lines; (2) high protein expression levels due to very high plastid DNA copy number per chloroplasts/cells/organ resulting in the accumulation of large amounts of the transgene's product in the chloroplast/ cell/organ; (3) possibility of multigene engineering (including cDNAs instead of full genes) through the use of transgene stacking in operons in a single transformation event; and (4) almost complete absence of pleiotropic effects due to subcellular compartmentalization of the transgene products (e.g., Staub et al 2000;Bock 2001Bock , 2013De Cosa et al 2001;Daniell et al 2002;Quesada-Vargas et al 2005;Verma and Daniell 2007;Oey et al 2009;Ruhlman et al 2010;Meyers et al 2010). From the biosafety point of view, the plastid technology (5) significantly increases transgene containment because plastids are maternally inherited in most crops, and therefore, the transgenes are not transmitted by pollen (Section 5) and outcrossing with weeds and other plants is not possible (Daniell et al 1998Daniell 2002;Hagemann 2004Hagemann , 2010.…”

“…In addition to carbohydrates, plastids are also involved in the synthesis and/or the storage of amino acids, lipids and fatty acids, starch, oil, and some secondary metabolites including carotenoids, terpenoids, alkaloids, lignanes, essential vitamins such as vitamin A, B1, B2, B3, B9, E, and K (Fitzpatrick et al 2012) or polyphenolic compounds like condensed tannins (Brillouet et al 2013) (reviewed in Verma and Daniell 2007;Solymosi and Keresztes 2012).…”