Promoters for sugarcane transformation : isolation of specific sequences and evaluation of rolC.
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Date
1997
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Abstract
Increasing the sucrose yield and the disease resistance of plants are two major
objectives of the transgenic sugarcane plant programme in South Africa. The
sugarcane culm has thus been identified as one of the main target areas for
transgene expression. A shortage of reliable promoter elements as well as patent
limitations have necessitated the isolation of promoters that are preferentially
expressed in the sugarcane culm. In the present study two different approaches were
followed to isolate such promoters, and the bacterial promoter, rolC, was evaluated for tissue-specific expression in sugarcane.
Differential display is a non-directed technique that was used to identify genes that
are differentially expressed in the mature sugarcane culm. The original method was
modified, and four putative culm-preferential fragments were isolated. Sequence and
hybridisation analyses revealed that these fragments were false positives, and could therefore not be used to obtain a culm-specific promoter.
Activity of the Agrobacterium rolC promoter was evaluated by analysing expression
patterns of two reporter genes in the mature culm of transgenic sugarcane plants.
Nucleic acid analyses indicated that the foreign DNA was incorporated into the sugarcane genome, and that mRNA transcripts were produced. Histochemical
analysis was done to visualise rolC-driven GUS and GFP expression in the mature
sugarcane culm. In both cases the reporter gene expression was restricted to the vascular bundles and specifically to the phloem.
A directed approach was followed to isolate the gene and subsequently the promoter
of the β-subunit of pyrophosphate-dependent phosphofructokinase (PFP-β). An
incomplete cDNA clone was obtained from a mature culm cDNA library, and was
used for the screening of a sugarcane genomic library. Two clones containing
different parts of the PFP-β gene were isolated. A Deletion Factory™ system was
used to analyse the clone containing the 5' end of the gene. The first five exons and
1747 bp of the 5' flanking region of the gene were sequenced. Preliminary activity analysis of the promoter region was done by constructing two expression vectors, and analysing transient GUS expression in sugarcane callus. Results indicated that the promoter is capable of driving foreign gene expression in callus. Transient expression levels were lower than that of the maize Ubi-1 promoter. Further analysis of the 5' flanking region will be done to establish whether cis-acting elements outside
the analysed area have an influence on the activity of the promoter.
Description
Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.
Keywords
Sugarcane., Sugarcane--Breeding., Andropogoneae., Sugarcane--Genetics., Sugarcane--Genetic engineering., Sugarcane--Research--South Africa., Theses--Botany.