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The isolation and molecular characterization of a 2S albumin gene from Picea glauca McInnis, Stephanie Marie
Abstract
2S albumins are a class of small seed storage proteins (SSPs) found widely among the Dicotyledonae and related to the prolamin SSPs of the Monocotyledonae. A genomic clone (PG2S) and related pseudogene with homology to the dicot 2S albumins were sequenced from the gymnosperm, Picea glauca. Northern blot analysis of developing spruce somatic embryos indicated embryo-specific expression of the conifer 2S albumin gene, in a pattern consistent with a seed storage protein. A translational fusion between 2.3 kb of the 5'-flanking region of PG2S and the uidA (β-glucuronidase) reporter gene was constructed to explore promoter function. Two distal deletions were also created, resulting in promoters deleted to a 5' position of -653 and -117, respectively, relative to the site of transcriptional initiation. The two larger constructs were used in the stable transformation of tobacco (Nicotiana tabacum cv. Xanthi) resulting in embryo-specific expression of the uidA gene in the developing tobacco embryo from long heart stage to embryo maturity with maximal expression in the torpedo stage embryo. Under control of the PG2S promoter GUS expression was not detected in any other tobacco tissues. There were no differences observed in pattern or strength of expression between the constructs in tobacco. The three PG2S promoter ; uidA reporter gene constructs were transiently expressed by microprojectile bombardment in a developmental series of interior spruce {Picea glauca/engelmannii) somatic embryos (proembryo, stage 2, early cotyledonary, mature and partially-dried mature), germinants and pollen. Transient expression of the 2.3 kb promoter construct mirrored 2S albumin mRNA levels in the proembryo, stage 2, early cotyledonary and mature embryo, as well as in germinants. In contrast, high levels of transient expression were observed in partially-dried mature embryos and in pollen, in which low and no 2S albumin mRNA was detected. The promoter deletions gave reduced levels of transient expression, but were not altered in seed-specificity. Putative regulatory motifs identified within the promoter were: ACGT, TGCA, CATG, CANNTG, and CCAC(C). Despite evolutionary distance, the 2S albumin promoter from spruce functions in a developmentally regulated, tissue-specific manner in an angiosperm; cis-elements and their co-ordinate transacting factors are conserved between gymnosperms and angiosperms.
Item Metadata
Title |
The isolation and molecular characterization of a 2S albumin gene from Picea glauca
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Creator | |
Publisher |
University of British Columbia
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Date Issued |
1998
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Description |
2S albumins are a class of small seed storage proteins (SSPs) found widely among the
Dicotyledonae and related to the prolamin SSPs of the Monocotyledonae. A genomic clone
(PG2S) and related pseudogene with homology to the dicot 2S albumins were sequenced from
the gymnosperm, Picea glauca. Northern blot analysis of developing spruce somatic embryos
indicated embryo-specific expression of the conifer 2S albumin gene, in a pattern consistent
with a seed storage protein. A translational fusion between 2.3 kb of the 5'-flanking region of
PG2S and the uidA (β-glucuronidase) reporter gene was constructed to explore promoter
function. Two distal deletions were also created, resulting in promoters deleted to a 5'
position of -653 and -117, respectively, relative to the site of transcriptional initiation. The
two larger constructs were used in the stable transformation of tobacco (Nicotiana tabacum
cv. Xanthi) resulting in embryo-specific expression of the uidA gene in the developing tobacco
embryo from long heart stage to embryo maturity with maximal expression in the torpedo
stage embryo. Under control of the PG2S promoter GUS expression was not detected in any
other tobacco tissues. There were no differences observed in pattern or strength of expression
between the constructs in tobacco.
The three PG2S promoter ; uidA reporter gene constructs were transiently expressed
by microprojectile bombardment in a developmental series of interior spruce {Picea
glauca/engelmannii) somatic embryos (proembryo, stage 2, early cotyledonary, mature and
partially-dried mature), germinants and pollen. Transient expression of the 2.3 kb promoter
construct mirrored 2S albumin mRNA levels in the proembryo, stage 2, early cotyledonary
and mature embryo, as well as in germinants. In contrast, high levels of transient expression
were observed in partially-dried mature embryos and in pollen, in which low and no 2S
albumin mRNA was detected. The promoter deletions gave reduced levels of transient
expression, but were not altered in seed-specificity.
Putative regulatory motifs identified within the promoter were: ACGT, TGCA,
CATG, CANNTG, and CCAC(C). Despite evolutionary distance, the 2S albumin promoter
from spruce functions in a developmentally regulated, tissue-specific manner in an angiosperm; cis-elements and their co-ordinate transacting factors are conserved between
gymnosperms and angiosperms.
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Extent |
14512332 bytes
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Genre | |
Type | |
File Format |
application/pdf
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Language |
eng
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Date Available |
2009-06-02
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Provider |
Vancouver : University of British Columbia Library
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Rights |
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.
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DOI |
10.14288/1.0088734
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URI | |
Degree | |
Program | |
Affiliation | |
Degree Grantor |
University of British Columbia
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Graduation Date |
1998-05
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Campus | |
Scholarly Level |
Graduate
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Aggregated Source Repository |
DSpace
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Item Media
Item Citations and Data
Rights
For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.