Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.
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"Since the main target of bromoxynil lies in the thylakoïd membrane of the chloroplast, we qucstionncd whcther the localisation of nitrilase inside the cell had an influence on bromoxynil resistance. We used the transit peptide of both the maize and sunflowcr SSU to target the nitrilase inside the chloroplast stroma (Schreier et al., 1985 ;Van den Broeck et al., 1985). The construction was made in such a way that the native enzyme should be rcleased in the stroma aftcr protcolytic cleavage at the cysteinc-mcthionine jonction bctween the transit peptide and the nitrilasc. "
[Show abstract][Hide abstract] ABSTRACT: Bromoxynil is an antidicot herbicide widely used on several cereal crops and with a short half life in the soil. A gene coding for a specific nitrilase which detoxifies this herbicide has been previously isolated (Stalker D.M. and Mc Bride K.E., 1987). The coding sequence of this gene was used to construct several chimaeric genes wich were introduced into industrial tobacco plants via Agrobacterium mediated transformation. We tested the possibility to use different promoters to obtain various patterns of expression of the detoxifying activity and were able to show that expression in the aerial parts of the plant was sufficient to achieve high levels of resistance. By using two types of transit peptides, we further tested the effect of the cellular enzyme localization, chloroplastic or cytoplasmic, on the resistance of these plants to bromoxynil. Resistance was observed both at the in vitro and at the mature plant level. Very low amounts of nitrilase were sufficient to efficiently protect the plants up to twenty times the lethal dose of herbicide. Resistance was stably inherited in the progeny in a mendelian fashion and field results confirm the high level of resistance with no alteration of the phenotype of these transgenic plants as compared with their non transgenic counterpart.
"Three cassettes that harboured a non-maize codon-optimized version (Chiu et al., 1996) were assembled under the control of either the 35S CaMV (Benfey and Chua, 1990), rice actin promoter , coupled with a 5¢-intron (Zhong et al., 1996) or the 1.7 kb maize C4 PepC promoter. (Yanagisawa and Izui, 1989) (ZmPpc1) The respective promoters were fused with translational enhancer element from the maize PPDK-A gene (Sheen, 1993), and GFP was targeted to plastids via the maize chloroplast RNA polymerase RpoTp transit peptide (Chang et al., 1999) for the 35S CaMV and rice actin cassettes, or the pea RBCS1 transit peptide (Van den Broeck et al., 1985; von Heijne et al., 1991) for the PEPC cassette. The non-codon-optimized GFP cassettes were subcloned into either the binary plasmid pPZP211 or pPZP212 (Hajdukiewicz et al., 1994), and the resultant vectors were referred to as pPTN343, pPTN372 and pPTN442, for the 35S, rice actin and PEPC promoters, respectively. "
[Show abstract][Hide abstract] ABSTRACT: Plastid number and morphology vary dramatically between cell types and at different developmental stages. Furthermore, in C4 plants such as maize, chloroplast ultrastructure and biochemical functions are specialized in mesophyll and bundle sheath cells, which differentiate acropetally from the proplastid form in the leaf base. To develop visible markers for maize plastids, we have created a series of stable transgenics expressing fluorescent proteins fused to either the maize ubiquitin promoter, the mesophyll-specific phosphoenolpyruvate carboxylase (PepC) promoter, or the bundle sheath-specific Rubisco small subunit 1 (RbcS) promoter. Multiple independent events were examined and revealed that maize codon-optimized versions of YFP and GFP were particularly well expressed, and that expression was stably inherited. Plants carrying PepC promoter constructs exhibit YFP expression in mesophyll plastids and the RbcS promoter mediated expression in bundle sheath plastids. The PepC and RbcS promoter fusions also proved useful for identifying plastids in organs such as epidermis, silks, roots and trichomes. These tools will inform future plastid-related studies of wild-type and mutant maize plants and provide material from which different plastid types may be isolated.
Full-text · Article · Feb 2010 · Plant Biotechnology Journal
"To prove this hypothesis, a protein was designed that can be targeted to both the chloroplast and the peroxisome. It is well known that the N-terminal transit peptide is necessary and sufficient for targeting a protein to the chloroplast (Van den Broeck et al., 1985; Smeekens et al., 1986; Bassham et al., 1991; Bruce, 2000; Lee et al., 2002). In contrast, the C-terminal three amino acid residues, SKL, are necessary and sufficient for targeting a protein to the peroxisome (Nito et al., 2002; Sparkes and Baker, 2002). "
[Show abstract][Hide abstract] ABSTRACT: One of the limiting factors in the production of recombinant proteins in transgenic plants is the low level of protein accumulation.
A strategy was investigated for a high level of protein accumulation in plant cells. A fungal xylanase encoded by XYLII of Trichoderma reesei was chosen as the model protein because xylanases have a high potential for applications in environment-related technologies.
Xylanase was expressed in the cytosol or targeted either to chloroplasts or peroxisomes alone, or to both organelles simultaneously.
When xylanase was targeted to both chloroplasts and peroxisomes simultaneously the amount of xylanase accumulated was 160%
of that in chloroplasts alone and 240% of that in peroxisomes alone although the transcript levels were similar among these
constructs. The growth stage of the transgenic plants also affected the total amount of xylanase; the highest level of accumulation
occurred at the time of flowering. This study provides genetic and biochemical data demonstrating that a high level of protein
accumulation in transgenic plants can be obtained by targeting a protein to both chloroplasts and peroxisomes at the same
Preview · Article · Feb 2006 · Journal of Experimental Botany