Infrequent transposition of Ac in lettuce, Lactuca sativa.
The maize transposable element Activator (Ac) is being used to develop a transposon mutagenesis system in lettuce, Lactuca sativa. Two constructs containing the complete Ac from the waxy-m7 locus of maize were introduced into lettuce and monitored for activity using Southern analysis and PCR amplification of the excision site. No transposition of Ac was detected in over 32 transgenic R1 plants, although these constructs were known to provide frequent transposition in other species. Also, no transposition was observed in later generations. In subsequent experiments, transposition was detected in lettuce calli using constructs that allowed selection for excision events. In these constructs, the neomycin phosphotransferase II gene was interrupted by either Ac or Ds. Excision was detected as the ability of callus to grow on kanamycin. Synthesis of the transposase from the cDNA of Ac expressed from the T-DNA 2' promoter resulted in more frequent excision of Ds than was observed with the wild-type Ac. No excision was observed with Ds in the absence of the transposase. The excision events were confirmed by amplification of the excision site by PCR followed by DNA sequencing. Excision and reintegration were also confirmed by Southern analysis. Ac/Ds is therefore capable of transposition in at least calli of lettuce.
Yang
C H
CH
Department of Vegetable Crops, University of California, Davis 95616.
Ellis
J G
JG
Michelmore
R W
RW
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004247:DNA; D004251:DNA Transposable Elements; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009713:Nucleotidyltransferases / Q000378:metabolism; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction; D019895:Transposases; D003313:Zea mays / Q000235:genetics*
D004251:DNA Transposable Elements; D004247:DNA; D009713:Nucleotidyltransferases; D019895:Transposases