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Efficient targeting of foreign genes into the tobacco plastid genome.

ABSTRACT: The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bombarded sample. The selected aadA gene and linked non-selectable genes cloned into the MCS are incorporated into the ptDNA by two homologous recombination events via the flanking ptDNA sequences. The transplastomes thus generated are stable, and are maternally transmitted to the seed progeny. The pPRV vector series targets insertions between the divergently transcribed trnV gene and the rps12/7 operon. The lack of readthrough transcription of appropriately oriented transgenes makes the vectors an ideal choice for the study of transgene promoter activity.

SUBMITTER: Zoubenko OV

PROVIDER: S-EPMC308375 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Efficient targeting of foreign genes into the tobacco plastid genome.

Zoubenko O V OV Allison L A LA Svab Z Z Maliga P P

Nucleic acids research 19940901 19

The pPRV plasmids are vectors for targeted insertion of foreign genes into the tobacco plastid genome (ptDNA). The vectors are based on the pUC119 plasmid which replicates in E. coli but not in plastids. The spectinomycin resistance (aadA) gene and a multiple cloning site (MCS) are flanked by 1.8-kb and 1.2-kb ptDNA sequences. Biolistic delivery of vector DNA, followed by spectinomycin selection, yields plastid transformants at a reproducible frequency, approximately 1 transplastomic line per bo ...[more]

PMID: 7937099

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Project description:BackgroundGenome editing by CRISPR/Cas9 has become a popular approach to induce targeted mutations for crop trait improvement. Soybean (Glycine max L. Merr.) is an economically important crop worldwide. Although gene editing has been demonstrated in soybean, its utilization in stably transformed plants through whole plant regeneration is still not widespread, largely due to difficulties with transformation or low mutation efficiencies.ResultsWe sought to establish a simple, efficient, and specific CRISPR/Cas9 system to induce heritable mutations in soybean through stable transformation. We targeted phytoene desaturase (PDS) genes due to the distinctive dwarf and albino phenotypes of the loss of function mutant. To evaluate gene editing efficiency and specificity, three constructs targeting each of the two homologous soybean PDS genes specifically, as well as two constructs targeting both simultaneously with one guide RNA were created. Instead of using cotyledonary nodes from germinated seedlings, we used 'half-seed' explants derived from imbibed seeds for Agrobacterium-mediated transformation of cultivar Williams 82. Transformed plants for all five constructs were recovered. Dwarf and albino phenotypes were observed in transgenic plants harboring the constructs targeting both PDS genes. Gene editing at the desired loci was detected in the majority of T0 transgenic plants, with 75-100% mutation efficiencies. Indel frequencies varied widely among plants (3-100%), with those exhibiting visible mutant phenotypes showing higher frequencies (27-100%). Deletion was the predominant mutation type, although 1-nucleotide insertion was also observed. Constructs designed to target only one PDS gene did not induce mutation in the other homologous counterpart; and no mutation at several potential off-target loci was detected, indicating high editing specificity. Modifications in both PDS genes were transmitted to T1 progenies, including plants that were negative for transgene detection. Strong mutant phenotypes were also observed in T1 plants.ConclusionsUsing simple constructs containing one guide RNA, we demonstrated efficient and specific CRISPR/Cas9-mediated mutagenesis in stably transformed soybean plants, and showed that the mutations could be inherited in progenies, even in plants that lost transgenes through segregation. The established system can be employed to edit other genes for soybean trait improvement.

Dataset Information

Efficient targeting of foreign genes into the tobacco plastid genome.

Publications

Efficient targeting of foreign genes into the tobacco plastid genome.

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