DNA insert from vector pHCW.T-7
Vector pHCW.T-7 contains four expression cassettes:
The SuRBHra expression cassette, intended to function as a
plant selectable marker, which is composed of:
* A constitutive fusion promoter EHS-Ubpp, comprising the
promoter/5ʹ untranslated region (UTR) of the epoxide hydrolase
(EHS) gene and the promoter/5ʹ UTR of the tetrameric ubiquitin
(Ubi) gene, both isolated from pineapple
* SuRBHra, a mutant acetolactate synthase (ALS) gene from tobacco
(Nicotiana tabacum)
* A transcription terminator sequence corresponding to the 3ʹ
untranslated region of the tobacco ALS gene
The BRIp-PSY-Ubpter expression cassette intended to express the
phytoene synthase protein, which is composed of:
* A modified promoter derived from the pineapple BRI gene
* The tangerine (Citrus unshiu) phytoene synthase (PSY) gene
* The transcription terminator sequence corresponding to the 3ʹ UTR
and flanking region of the pineapple Ubi gene
The BRIp-bLcy RNAi-Ubpter expression cassette, intended to
suppress expression of the pineapple lycopene β-cyclase protein,
which is composed of:
* A modified promoter derived from the pineapple BRI gene
* A partial coding sequence of pineapple bLcy in the sense
orientation,
* The intron 2 sequence of the potato (Solanum tuberosum) ST-LSI
gene,
* A partial coding sequence of pineapple bLcy in the anti-sense
orientation
* The transcription terminator sequence corresponding to the 3ʹ UTR
and flanking region of the pineapple Ubi gene
The BRIp-eLcy RNAi-Ubpter expression cassette, intended to
suppress expression of the pineapple lycopene ε-cyclase protein,
which is composed of:
* A modified promoter derived from the pineapple BRI gene
* A partial coding sequence of pineapple eLcy in the sense
orientation
* The intron 2 sequence of the potato (S. tuberosum) ST-LSI
gene
* * A partial coding sequence of pineapple eLcy in the anti-sense
orientation
The transcription terminator sequence corresponding to the 3ʹ UTR
and flanking region of the pineapple Ubi gene
DNA insert from vector pHCWflACC3ʹ-2
Vector pHCWflACC3ʹ-2 contains two expression cassettes:
The SuRBHra expression cassette described above, intended as a
plant selectable marker
The Ubpp-flACC3ʹ RNAi-Upbter expression cassette, intended to
suppress expression of 1-aminocyclopropane-1-carboxylic acid
synthase (ACS), which is composed of:
* A promoter sequence corresponding to the promoter/5ʹ region of
the pineapple Ubi gene
* A partial coding sequence of pineapple meristem ACS gene flACC3ʹ
in the sense orientation
* The intron 2 sequence of the potato (S. tuberosum) ST-LSI
gene
* A partial coding sequence of pineapple meristem ACS gene flACC3ʹ
in the anti-sense orientation
* The transcription terminator sequence corresponding to the 3ʹ UTR
and flanking region of the pineapple Ubi gene.
Characteristics of the introduced DNA
Southern blot analysis was carried out to determine the
characteristics and stability of the introduced DNA. Studies
provided data that was consistent with four complete integrations
of the pHCW.T-7 T-DNA, one irregular integration of the
pHCWflACC3ʹ-2 T-DNA, and three partial integrations of the
pHCWflACC3ʹ-2 right border region.
Furthermore, the data indicated that at least one intact copy of
pHCW.T-7 is present in the EF2-114 genome, in addition to
rearranged copies of pHCW.T-7, the exact structure of which could
not be completely elucidated. In addition the data supports that no
intact copies of pHCWflACC3ʹ-2 are present in EF2-114, and that
extensive rearrangements of pHCWflACC3ʹ-2 genetic elements had
occurred.
Copy number analysis was consistent with the integration of four
copies of the pHCW.T-7 plasmid, of which two were complete. There
was also evidence of one partial integration of the pHCWflACC3ʹ-2
plasmid.
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