Chloroplast targeting signal regulates transgene expression in rice

Jang, In‐Cheol; Lee, K. H.; Nahm, Baek Hie; Kim, J. K.; Khush, G. S.; Brar, D. S.; Hardy, B.

doi:10.1142/9789812814319_0151

Cited by 10 publications

(13 citation statements)

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“…The RbcS3 promoter was highly active in leaf tissues especially in the presence of its functional transit peptide sequence TP3. These results are consistent with results of RbcS1 and TP1 where TP1 enhanced the levels of transcripts significantly (Jang et al 2002). Furthermore, transient expressions of constructs, 35S:TP3:GFP and 35S:TP1:GFP in rice leaf protoplasts Fig.…”

Section: Discussionsupporting

confidence: 91%

“…Two transgenic lines harboring RbcS3:TP3:GFP and RbcS3:GFP were analyzed in comparison with the control transgenic lines. The RbcS1:TP1:GFP and RbcS1:GFP (Jang et al 2002) are positive controls for RbcS3:TP3:GFP and RbcS3:GFP, respectively. PGD1 (Park et al 2010) and ZmUbi1 (Cornejo et al 1993) are known constitutive gene promoters.…”

Section: Discussionmentioning

confidence: 99%

“…The correct cleavage site of TP:GFP is between the cysteine (residue 47) and methionine (residue 48) during transport to chloroplast of the reporter gene in transgenic plants (Jang et al 1999). The high level of GFP protein accumulation, primarily reflected in the increase of the mRNA levels, was due to the presence of the functional RbcS1 transit peptide (Jang et al 2002). Furthermore, the paralogs of RbcS genes have highly conserved coding regions although the levels and patterns of expression can vary.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

Park

Jeong

Choi

et al. 2015

Plant Biotechnol Rep

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Here, we report characterization of an RbcS promoter and its transit peptide for use in chloroplast-targeted expression. RbcS3 that encodes a small subunit of ribulose bisphosphate carboxylase/oxygenase is of great interest due to its green tissue-specific expression such as leaf and leaf sheaths. We isolated the RbcS3 promoter and its transit peptide sequence and linked to GFP for characterization in transgenic plants. Our qRT-PCR analysis on leaves and roots of transgenic plants revealed that GFP transcript levels were higher by fivefold in leaves of RbcS3:TP3:GFP transgenic lines than in those of the RbcS3:GFP transgenic lines, suggesting that the transcript levels were increased by chloroplast-targeted expression, as shown in previously characterized RbcS1:TP1 system. The transit peptide sequence TP3 was sufficient to target GFP to chloroplasts as observed in protoplasts of RbcS3:TP3:GFP transgenic lines. GFP fluorescence in RbcS3:GFP plants was detected only in the cytosol. The GFP protein levels in leaves of RbcS3:TP3:GFP plants were 0.12-0.23 % of the total soluble leaf proteins. These levels were higher or comparable to those of PGD1:GFP plants (GFP driven by a constitutive promoter PGD1). Taken together, these results indicate that the fusion of the RbcS3 promoter to its transit peptide, RbcS3:TP3, provides a chloroplast-targeting expression system.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

Park

Jeong

Choi

et al. 2015

Plant Biotechnol Rep

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“…For this purpose, a rbcS promoter from desi cotton (G. arboreum) was isolated. The small subunits promoters and their transit peptides are attractive candidates for expression of genes at high levels and have shown promising expression results in driving the expression of introduced genes (Wong et al 1992;Fujimoto et al 1993;Data et al 1998;Song et al 2000;Jang et al 2002;Liu et al 2005;Panguluri et al 2005;Amarasinghe et al 2006;Anisimov et al 2007;Suzuki et al 2009;Kim et al 2009 andBakhsh et al 2011a, b). rbcS promoter was cloned in a plant expression vector pCAMBIA 1301.…”

Section: Discussionmentioning

confidence: 99%

A molecular approach to combat spatio-temporal variation in insecticidal gene (Cry1Ac) expression in cotton

2011

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Spatio-temporal expression of an insecticidal gene (Cry1Ac) in pre existing transgenic lines of transgenic cotton was studied. Seasonal decline in expression of Cry1Ac differed significantly among different cotton lines tested in the field conditions. The leaves of the Bt cotton plants were found to have the highest levels of toxin expression followed by squares, bolls, anthers and petals. Expression of the gene decreased consistently with the age of plants. Toxin expression in fruiting parts was not enough to confer full resistance against bollworms. The reduction in efficacy of transgenic cotton plants late in the season was attributed to reduction in promoter activity. For this purpose, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) promoter was isolated from Gossypium arboreum that was further cloned upstream of an insecticidal gene (Cry1Ac) in expression vector pCAMBIA 1301. A local cotton cultivar NIAB-846 was transformed with Cry1Ac driven by rbcS promoter. The same cotton cultivar was also transformed with Cry1Ac gene driven by 35SCaMV promoter to compare the expression pattern of insecticidal gene under two different promoters. The results showed that rbcS is an efficient promoter to drive the expression of Cry1Ac gene consistent throughout the life of cotton plant as compared to 35S promoter. The use of tissue specific promoter is also useful for addressing the biosafety issues as the promoter activity is limited to green parts of plants, hence no gene expression in roots, cotton seed and other cotton products and by products.

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“…Previously, we observed that the targeting of foreign gene products to plastids using the rice rbcS-tp sequence increased the protein product levels in transgenic rice plants (Jang et al 1999(Jang et al , 2002. In our current study, a synthetic truncated cry1Ac was linked to the rice rbcS-tp sequence for chloroplast-targeted expression.…”

Section: Introductionmentioning

confidence: 60%

Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection

Kim

Suh

Park

et al. 2009

Planta

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To increase insect resistance in transgenic rice plants, a synthetic truncated cry1Ac gene was linked to the rice rbcS promoter and its transit peptide sequence (tp) for chloroplast-targeted expression. Several transgenic lines were generated by the Agrobacterium-mediated transformation method and the expression levels of the transgene were compared with untargeted expression. Use of the rbcS-tp sequence increased the cry1Ac transcript and protein levels by 25- and 100-fold, respectively, with the accumulated protein in chloroplasts comprising up to 2% of the total soluble proteins. The high level of cry1Ac expression resulted in high levels of plant resistance to three common rice pests, rice leaf folder, rice green caterpillar, and rice skipper, as evidenced by insect feeding assays. Transgenic plants were also evaluated for resistance to natural infestations by rice leaf folder under field conditions. Throughout the entire period of plant growth, the transgenic plants showed no symptoms of damage, whereas nontransgenic control plants were severely damaged by rice leaf folders. Our results demonstrate that the targeting of cry1Ac protein to the chloroplast using the rbcS:tp system confers a high level of plant protection to insects, thus providing an alternative strategy for crop insect management.

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Chloroplast targeting signal regulates transgene expression in rice

Cited by 10 publications

References 0 publications

Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

Characterization of the rice RbcS3 promoter and its transit peptide for use in chloroplast-targeted expression

A molecular approach to combat spatio-temporal variation in insecticidal gene (Cry1Ac) expression in cotton

Chloroplast-targeted expression of synthetic cry1Ac in transgenic rice as an alternative strategy for increased pest protection

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