Ac-mediated trans-activation of the Ds element in rice (Oryza sativa L.) cells as revealed by GUS assay.
A method using particle bombardment and beta-glucuronidase (GUS) assay was applied to rice callus for detecting the excision of the maize Ds element trans-activated by the Ac transposase source. Excision of Ds biolistically introduced into rice callus resulted in the restoration of the interrupted gus gene expression, allowing visual detection of trans-activation two days after bombardment. Only the transgenic callus lines expressing the Ac transposase gene and the wild-type callus co-transformed with Ac and Ds revealed GUS activity. Frequency of excision, estimated based on the relative GUS activity, ranged from 0.3% to 2.2%. Callus lines showing different levels of Ac transcripts revealed varying excision frequencies. At the later stages of callus growth after selection for the Ac/Ds transformed lines, excision events were detected by GUS assay and confirmed by PCR and sequence analyses of the excision sites in individual colonies. GUS activity was also demonstrated in the primary regenerants from the Ac/Ds-transformed callus colonies. The method described in this study may be used as an approach for rapid detection of excision events and assessment of various factors limiting Ac/Ds activity in rice cells.
Solis
R
R
Graduate School of Science and Technology, Faculty of Agriculture, Kobe University, Japan.
Takumi
S
S
Mori
N
N
Nakamura
C
C
D019470:Biolistics; D002478:Cells, Cultured; D004251:DNA Transposable Elements / Q000235:genetics*; D018744:DNA, Plant / Q000235:genetics / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D005813:Genes, Synthetic; D005966:Glucuronidase / Q000096:biosynthesis* / Q000235:genetics; D012275:Oryza / Q000235:genetics*; D010940:Plant Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D015533:Transcriptional Activation; D019895:Transposases / Q000378:metabolism*
D004251:DNA Transposable Elements; D018744:DNA, Plant; D004274:DNA, Recombinant; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D019895:Transposases; D005966:Glucuronidase