Molecular cloning, immunolocalization, and transgenic expression in yeast and tobacco of three cDNAs that encode sugar transporters in sugar beet
Chiou, Tzyy-Jen
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Permalink
https://hdl.handle.net/2142/21331
Description
Title
Molecular cloning, immunolocalization, and transgenic expression in yeast and tobacco of three cDNAs that encode sugar transporters in sugar beet
Author(s)
Chiou, Tzyy-Jen
Issue Date
1995
Doctoral Committee Chair(s)
Bush, Daniel R.
Department of Study
Biology
Discipline
Biology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Biology, Botany
Biology, Plant Physiology
Language
eng
Abstract
Sugar transporter genes from sugar beet have been cloned as a first step in understanding their contribution to assimilate partitioning. Two cloning strategies were attempted. One was functional complementation of a yeast mutant with a sugar beet cDNA library constructed in a yeast expression vector. The other approach was PCR amplification based on sequence homologies found in a superfamily of sugar transporters. No promising clones were isolated by functional complementation. In contrast, several cDNA clones have been obtained by PCR amplification and subsequent screening of a cDNA library.
Three full length cDNA clones (cDNA-1, -205, -397) and one partial clone (PCR81) encoding putative sugar transporters were identified. The deduced amino acid sequences for cDNA-205 and -397 are the same except for a few residues at the C terminus. The estimated molecular weights of the three full length clones are between 54 and 60 kDa. As judged by sequence homologies (20-30% amino acid identity and 60-70% similarity with other sugar carriers) and hydropathy profiles, these clones are members of the sugar transporter gene family.
Northern analysis showed differential and tissue specific expression patterns for these putative sugar transporters. Southern analysis suggests that there are multiple copies of cDNA-1 and cDNAs-205/397 in the sugar beet genome. The proteins encoded by these putative sugar transporter genes were also visualized on SDS-PAGE following in vitro transcription and translation with microsome vesicles.
In order to understand the biological functions of these putative sugar transporters, their cognate cDNAs were expressed in transgenic yeast and tobacco. In addition, polyclonal antibodies were generated against each protein using E. coli over-expression and synthetic peptides. A 40 kDa polypeptide, similar to the in vitro translation product, was identified in cDNA-1 transformed yeast, by the antibodies directed against either the N or C terminal peptide. However, this protein could not be detected by antibodies directed against the central hydrophilic domain. The same protein was also observed in light membrane fractions of sugar beet and transgenic tobacco. Linear sucrose gradients and flotation centrifugation revealed that the cDNA-1 protein is located in the tonoplast membrane. This is the first member of sugar transport family targeted to the vacuole. Unfortunately, I was not able to identify its transport substrate by using membranes isolated from transformed yeast and tobacco.
Although the transported substrates of these putative sugar transporters have yet to be identified, I believe each makes a unique contribution to assimilate partitioning, an essential process for plant growth and development.
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