Chloroplast targeting of neomycin phosphotransferase II with a pen transit peptide in electroporated barley mesophyll protoplasts

Teemu Teeri, Geeta Patel, Kristian Aspegren, Veli Kauppinen

Research output: Contribution to journalArticleScientificpeer-review

25 Citations (Scopus)

Abstract

Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.
Original languageEnglish
Pages (from-to)187-190
JournalPlant Cell Reports
Volume8
Issue number4
DOIs
Publication statusPublished - 1989
MoE publication typeA1 Journal article-refereed

Fingerprint

kanamycin kinase
mesophyll
protoplasts
chloroplasts
barley
peptides
electroporation
ribulose-bisphosphate carboxylase
genes
peas
gene transfer
Hordeum vulgare
polypeptides
proteinases
promoter regions
cells
enzyme activity
synthesis
DNA

Cite this

Teeri, Teemu ; Patel, Geeta ; Aspegren, Kristian ; Kauppinen, Veli. / Chloroplast targeting of neomycin phosphotransferase II with a pen transit peptide in electroporated barley mesophyll protoplasts. In: Plant Cell Reports. 1989 ; Vol. 8, No. 4. pp. 187-190.
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abstract = "Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.",
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Chloroplast targeting of neomycin phosphotransferase II with a pen transit peptide in electroporated barley mesophyll protoplasts. / Teeri, Teemu; Patel, Geeta; Aspegren, Kristian; Kauppinen, Veli.

In: Plant Cell Reports, Vol. 8, No. 4, 1989, p. 187-190.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Chloroplast targeting of neomycin phosphotransferase II with a pen transit peptide in electroporated barley mesophyll protoplasts

AU - Teeri, Teemu

AU - Patel, Geeta

AU - Aspegren, Kristian

AU - Kauppinen, Veli

PY - 1989

Y1 - 1989

N2 - Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.

AB - Routine methods for stable gene transfer to cereals are not yet available. To be able to study chloroplast targeting in barley (Hordeum vulgare L.) cells, the expression of recombinant genes was assayed in barley mesophyll protoplasts after electroporation of DNA. The CaMV 35S transcript promoter was attached to a chimeric gene consisting of a pea RuBisCo small subunit transit peptide coding sequence (tp) and the gene coding for neomycin phosphotransferase II (nptII). As a control, a construction with no transit peptide coding segment was used. 48 hours after electroporation, a fraction of the protoplasts was lysed and intact chloroplasts were isolated. Protoplasts electroporated with either of the gene constructions showed strong NPTII activity. However, enzyme activity was detected in chloroplasts only when thetp-nptII gene construction was used. Protease treatment of the chloroplasts confirms that the pea RuBisCo small subunit transit peptide is targeting the NPTII polypeptide into the chloroplasts, subsequent to the synthesis of the hybrid precursor in barley cells.

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SN - 0721-7714

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