Cloning and expression of sesquiterpene synthase genes from lettuce (Lactuca sativa L.).
Sesquiterpenoid lactones (SLs) from lettuce (Lactuca sativa L.) include constitutive components of latex such as lactucin and the induced phytoalexin, lettucenin A. A redundant primer strategy was used to recover two full length cDNA clones (LTC1 and LTC2) encoding sesquiterpene synthases from a cDNA library derived from seedlings with the red spot disorder, which accumulate phytoalexins. Recombinant enzymes produced from LTC1 and LTC2 in Escherichia coli catalysed the cyclisation of farnesyl diphosphate to germacrene A, potentially an early step in the biosynthesis of SLs. RT-PCR analysis showed LTC1 and LTC2 were expressed constitutively in roots, hypocotyls and true leaves but not in cotyledons. Expression in cotyledons was induced by challenge with the downy mildew pathogen Bremia lactucae in the disease resistant cultivar Diana. Southern hybridisation experiments showed that LTC1 and LTC2 were not part of a multigene family. The germacrene A synthases provide targets for modified expression to generate beneficial modifications to the SL profile in lettuce.
Bennett
Mark H
MH
Department of Agricultural Sciences, Imperial College, Wye, Ashford, Kent TN25 5AH, UK.
Mansfield
John W
JW
Lewis
Mervyn J
MJ
Beale
Michael H
MH
D000595:Amino Acid Sequence; D019755:Carbon-Carbon Lyases / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D019295:Computational Biology; D017931:DNA Primers; D015870:Gene Expression; D018545:Lactuca / Q000201:enzymology* / Q000235:genetics / Q000382:microbiology; D008969:Molecular Sequence Data; D009868:Oomycetes / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D011994:Recombinant Proteins; D019755:Carbon-Carbon Lyases; C051130:trichodiene synthetase