Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco

Jeong, Seok-Won; Jeong, Won Joong; Woo, Janghee; Choi, Dong-Woog; Park, Youn‐Il; Liu, J. R.

doi:10.1007/s00299-003-0740-4

Cited by 26 publications

(21 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…A dicistronic construct was built to avoid having duplicated rrn promoter sequences in the expression cassette. Dicistronic constructs have been shown to generate significant levels of recombinant protein expression in tobacco plastids (Daniell et al, 1998, De Cosa et al, 2001, Lee et al, 2003, Jeong et al, 2004. The HA gene is preceeded by an 18 bp synthetic leader that includes a Shine-Dalgarno-like sequence (typically GGAGG) that is required for translation (Svab and Maliga, 1993;Hirose and Sugiura 2004).…”

Section: Resultsmentioning

confidence: 99%

“…The LEC1 dicistronic cassette contains the aadA gene and the HA gene under the control of the lettuce rrn operon promoter (PLs-rrn). A similar dicistronic format was used to co-express aadA with various other transgenes in the tobacco plastid including gfp (Jeong et al, 2004), 5-enol-pyruvate shikimate-3-phosphate synthase (Daniell et al, 1998), the Cry2Aa2 operon (De Cosa et al, 2001), and a trehalose phosphate synthase gene (Lee et al, 2003). Although fertile, homoplasmic, plastidtransformed plant lines were obtained, only mRNA, and no HA protein, was detected in these lines (data not shown), even though HA protein was detected at a high level in E. coli containing this construct (data not shown).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

et al. 2005

View full text Add to dashboard Cite

Although plastid transformation in higher plants was first demonstrated in the early 1990s it is only recently that the technology is being extended to a broader range of species. To date, the production of fertile transplastomic plants has been reported for tobacco, tomato, petunia, soybean, cotton and Lesquerella fendleri (Brassicaceae). In this study we demonstrate a polyethylene glycol-mediated plastid transformation system for lettuce that generates fertile, homoplasmic, plastid-transformed lines. Transformation was achieved using a vector that targets genes to the trnA/trnI intergenic region of the lettuce plastid genome employing the aadA gene as a selectable marker against spectinomycin. Spectinomycin resistance and heterologous gene transcription were shown in T 1 plants derived from self-pollinated primary regenerants demonstrating transmission of the plastid-encoded transgene to the first seed generation. Crossing with male sterile wild-type lettuce showed that spectinomycin resistance was not transmitted via pollen. Constructs containing the gfp gene showed plastid-based expression of green fluorescent protein. The lettuce plastid could have potential both as a production and a delivery system for edible human therapeutic proteins.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

et al. 2005

View full text Add to dashboard Cite

show abstract

“…e use of spectinomycin-resistant aadA as a selectable marker markedly increased the transformation efficiency to 1-2.5 transformants per plate (Svab and Maliga 1993;Khan and Maliga 1999). Despite the difference in vector construction, such as homologous regions and selectable marker genes, plastid transformants were produced not only in tobacco (Daniell et al 1998;Bock et al 1996;Jeong et al 2004) but also in other species (Day and GoldschmidtClermont 2011;Cui et al 2011). However, plastid transformation e ciency in other species except tobacco was still very low, making it difficult to obtain one plastid transformant per bombarded plate.…”

mentioning

confidence: 97%

Improvement of the plastid transformation protocol by modifying tissue treatment at pre- and post-bombardment in tobacco

Okuzaki

Tabei

2012

Plant Biotechnology

View full text Add to dashboard Cite

Plastid transformation in higher plants has been established in tobacco using particle bombardment, and the protocol has been used as a model in other plant species; however, as target materials, tobacco leaves may be partially exposed to unexpected abiotic stresses-drought stress during post-bombardment culture and damage due to cutting before selection culture. Plastid transformation e ciency may be increased by modi cation of leaf treatment to lessen such damage. In the modi ed protocol, tobacco leaves were cut into pieces (5×5 mm) and placed on the culture medium plate (approximately y pieces per plate) for one day before bombardment. A er bombardment, these pieces were transferred onto the non-selection medium and cultured for three days; they were then transferred onto spectinomycin selection culture medium. A transformation vector containing an aminoglycoside 3′-adenylyltransferase gene and a green uorescence protein gene were used for plastid transformation. Approximately four independent plastid transformants per bombarded plate were obtained on average using the modi ed protocol; the transformation e ciency was 1.6 times higher than that in a control experiment using the standard protocol. Modi ed leaf treatment improved the e ciency and stability of plastid transformation. is nding should aid plastid transformant production in tobacco and other plant species.

show abstract

“…These include the prevention of gene flow to other plants due to maternal inheritance (Daniell 2002), a lack of gene silencing, and positional effects because integration of the foreign DNA is targeted instead to the precise region without disrupting unwanted genes (de Cosa et al 2001;Daniell et al 2002;Lee et al 2003) and the insertion of multiple heterologous genes or operons under the control of a single regulatory sequence (de Cosa et al 2001;Jeong et al 2004). It is also useful for studies with reverse genetics of the functioning of unknown genes, ORFs, or sequences of a plastid genome that use targeted disruption.…”

mentioning

confidence: 99%

Stable Plastid Transformation in Nicotiana benthamiana

et al. 2009

View full text Add to dashboard Cite

Plastids from Nicotiana benthamiana were transformed with the vector for dicistronic expression of two genes-aminoglycoside 3'-adenyltransferase (aadA) and green fluorescent protein (gfp)-in the plastids of Nicotiana tabacum. Transplastomic shoots exhibited green fluorescence under UV light. Transformation efficiencies were similar between species. Although the border sequence (trnI and trnA) for homologous recombination to transform the plastid genome of N. benthamiana was identical to that sequence of N. tabacum, the exception was a 9-bp addition in the intron of trnI. This indicated that the N. tabacum sequence used as a border region for recombination was sufficient to insert the foreign gene into the target site between the trnI and trnA of N. benthamiana with similar efficiency. Southern blot analysis detected the presence of aadA and gfp between trnI and trnA in the plastid genome of N. benthamiana. Northern and western blot analyses revealed high expression of gfp in the plastids from petals and leaves. Our results suggest that the plastid transformation system established here is applicable to investigations of the interactions between plastid and nucleus in N. benthamiana.

show abstract

Dicistronic expression of the green fluorescent protein and antibiotic resistance genes in the plastid for selection and tracking of plastid-transformed cells in tobacco

Cited by 26 publications

References 8 publications

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

Stable Plastid Transformation in Lettuce (Lactuca sativa L.)

Improvement of the plastid transformation protocol by modifying tissue treatment at pre- and post-bombardment in tobacco

Stable Plastid Transformation in Nicotiana benthamiana

Contact Info

Product

Resources

About