The pKYLX series of plant gene expression vectors

We have assembled a series of expression vectors for use with Agrobacterium-mediated and biolostic gene transfer in plants. The Agrobacterium-based plasmids (pKYLX5, pKYLX7, pKYLX71, and pKYLX71:35S2) all use, as a "shell", the pGA472 plasmid described by An et al. (1); the construction and basic characterization of pKYLX5 and pKYLX7 have been described (2). The biolistic plasmid (pKYLX80) contains the expression cassette and kanamycin resistance gene from pKYLX71:35S2, but in a slightly modified pBluescript background; this permits the production of large quantities of these plasmids.

Illustrations of these plasmids follow, as does the sequence of the expression cassette of pKYLX71:35S2.

Go to:

  • pKYLX71 map
  • pKYLX80 map
  • pKYLX expression cassette sequence

    • pKYLX71

    Key: Restriction enzyme sites are as shown.
    TL, TR - left and right T-DNA borders (see reference 2 for a detailed description of their origin).
    The multiple cloning site for pKYLX71 and pKYLX71:35S2 (these are the plasmids currently in use) is: HindIII*-BamHI-XhoI*-PstI-SacI*-XbaI* (* - unique sites).
    The promoters that have been described are:

    in pKYLX5 - the pea rbcS-E9 promoter
    in pKYLX7 and pKYLX71 - the CaMV 35S promoter
    in pKYLX71:35S2 - a modified 35S promoter with a duplicated "enhancer" region

    Arrows show directions of transcription for the expression cassette and kanamycin resistance gene.
    This plasmid confers tetracycline (12.5 µg/ml) and kanamycin (50 µg/ml) resistance upon E. coli and Agrobacterium tumefaciens. The kanamycin resistance gene is effective at kanamycin concentrations as high as 500 µg/ml in tobacco. These plasmids have been used in several labs, and work well in Arabidopsis.

    pKYLX80

    Key: BstEII*, BstXI* - self explanatory
    S - SalI*
    P - PstI
    B - BamHI
    C - ClaI*
    E - EcoRI*
    mcs - multiple cloning sites (HindIII*-BamHI-XhoI*-PstI-SacI*-XbaI*; * - unique sites)
    TR - nos T-DNA right border

    The arrow shows the direction of transcription for the expression cassette. The orientation of the kanamycin resistance gene is the same as described for pKYLX71.

    This plasmid confers ampicillin (50-200 µg/ml) and kanamycin (50 µg/ml) resistance upon E. coli and Agrobacterium tumefaciens. The kanamycin resistance gene is effective at kanamycin concentrations as high as 500 µg/ml in tobacco.

    The sequence of the expression cassette in pKYLX71:35S2 and pKYLX80


    GAATTCGCCCGGGGATCTCCTTTGCCCCAGAGATCACAATGGACGACTTCCTATATCT
    CTACGATCTAGTCAGGAAGTTCGACGGAGAAGGTGACGATACCATGTTCACCACTG
    ATAATGAGAAGATTAGCCTTTTCAATTTCAGAAAGAATCCTAACCCACAGATGGTTA
    GAGACGCTTACGCAGCAGGTCTCATCAAGACGATCTACCCGAGCAATAATCTCCAG
    GAGATCAAATACCTTCCCAAGAAGGTTAAAGATGCAGTCAAAAGATTCAGGACTAA
    CTGCATCAAGAACACAGAGAAAGATATATTTCTCAAGATCAGAAGTACTATTCCAGT
    ATGGACGATTCAAGGCTTGCTTCACAAACCAAGGCAAGTAATAGAGATTGGAGTCT
    CTAAAAAGGTAGTTCCCACTGAATCAAAGGCCATGGAGTCAAAGATTCAAATAGAG
    GACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACG
    ACTCAATGACAAGAAGAAAATCTTCGTC{AACATGGTGGAGCACGACACGCTTGTCT
    ACCTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGAATTGAGACTTTT
    CAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCAC
    TTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGAT
    AAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACC
    CCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGC
    AAGTGGATTGATGTGAT    }AACATGGTGGAGCACGACACGCTTGTCTACCTCCAAAAA
    TATCAAAGATACAGTCTCAGAAGACCAAAGGGAATTGAGACTTTTCAACAAAGGGT
    AATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAG
    ATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGC
    CATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAG
    GAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGAT
    GTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACC
    CTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGAAATCACCAGT
    CTCTCTCTAAGCTTGGATCCTCGAGCTGCAGGAGCTCGAATTGATCCTCTAG
    A    GCTTTCGTTCGTATCATCGGTTTCGACAACGTTCGTCAAGTTCAATGCATCAGTTTC
    ATTGCGCACACACCAGAATCCTACTGAGTTCGAGTATTATGGCATTGGGAAAACTGT
    TTTTCTTGTACCATTTGTTGTGCTTGTAATTTACTGTGTTTTTTATTCGGTTTTCGCTATC
    GAACTGTGAAATGGAAATGGATGGAGAAGAGTTAATGAATGATATGGTCCTTTTGTT
    CATTCTCAAATTAATATTATTTGTTTTTTCTCTTATTTGTTGTGTGTTGAATTTGAAATT
    ATAAGAGATATGCAAACATTTTGTTTTGAGTAAAAATGTGTCAAATCGTGGCCTCTA
    ATGACCGAAGTTAATATGAGGAGTAAAACACTTGTAGTTGTACCATTATGCTTATTC
    ACTAGGCAACAAATATATTTTCAGACCTAGAAAAGCTGCAAATGTTACTGAATACAA
    GTATGTCCTCTTGTGTTTTAGACATTTATGAACTTTCCTTTATGTAATTTTCCAGAATCC
    TTGTCAGATTCTAATCATTGCTTTATAATTATAGTTATACTCATGGATTTGTAGTTGAG
    TATGAAAATATTTTTTAATGCATTTTATGACTTGCCAATTGATTGACAACATGCATCA
    ATCGAT

    Notes: the region duplicated to create the 35S2 promoter is bounded by brackets and italicized (this is the first of the two repeats), and the transcription start site, multiple cloning sites and the principal 3' end in the rbcS-E9 3' region are set off in bold type. The expression cassette is bounded by Eco RI and Cla I sites.

    References

    1. Schardl, C., Byrd, A. D., Benzion, G. B., Altschuler, M. A., Hildebrand, D. F., and Hunt, A. G. (1987). Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61, 1-11.

    2. An, G., Watson, B. G., Stachel, S., Gordon, M. P., and Nester, E. W. (1985) New cloning vehicles for transformation of higher plants. EMBO J. 4, 277-284.

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    For more information, contact Arthur Hunt (aghunt00@pop.uky.edu)