Cloning and molecular characterization of vffl gene coding forisomes of vicia faba

Forisomes are phloem-specific proteins of the family of the Fabaceae which undergo a rapid, reversible and ATP-independent comformational change when stimulated by variations in Ca2+ levels or pH. These proteins, present in the sieve elements, switch instantaneously between a dispersed and a condensed conformation, allowing them to control the stream of photo-assimilates in the phloem after wounding by sealing the conducting tubes. Isolation of Forisomes from Vicia faba permitted the sequencing of peptides from which specific oligonucleotides were designed for library screening. In addition, polyclonal Forisome-specific antibodies were obtained. First attempts to identify Forisome-encoding genes were performed by hybridization screening of subtracted and non-subtracted Vicia faba derived cDNA libraries, however, no successful results could be retrieved. In an alternative approach, immunoscreening along with PCR screening of cDNA expression library of Vicia faba plant tips was carried out. Neither the identification of cDNAs encoding antigenic Forisome fusion proteins nor the allocation of DNA- and/or DNA fragments to the Forisome genes was possible. Aligning the isolated Forisome peptide sequences from Vicia faba with a recently published EST-library for Medicago truncatula – the model plant for Fabaceae – enabled the identification of patches of amino acids conserved among the two species belonging to the same DNA sequence. This important finding provided the basis for the isolation of genes encoding Forisome proteins. Hence, the rapid amplification of cDNA ends (RACE) together with genome walking was used to obtain the full-length cDNA as well as the genomic sequence of the Forisome gene vff1. Sequence analysis of the amplified vff1 was performed and even thought no well-known Ca2+-binding motif could be identified, different domains which might be involved in Ca2+ dependent reaction of the Forisomes were recognized within the vff1 gene. Cloning and recombinant expression of the isolated vff1 gene in E. coli permitted the generation of VFF1-specific polyclonal antibodies. Immunological evidence of the isolated vff1 gene encoding a Forisome protein was obtained by means of western blot analysis and confocal immunofluorescence techniques. Further experiments were performed with the aim of identifying and cloning the vff1 gene 5'-flanking region. The putative promoter of the vff1 gene was isolated from genomic DNA by genome walking and its activity as well as tissue specificity was confirmed in transgenic tobacco plants using GUS as a reporter system. Sequence analysis revealed two conserved motifs shared with several previously reported phloem-specific promoters. Taken together, these results indicate that the 5'-upstream region of vff1 functions as a promoter (designated vff1P) that directs preferential expression of the uidA gene to the phloem of transgenic tobacco plants. Besides the tissue-specificity a spatial and temporal regulation of vff1P was observed during development of transgenic tobacco seedlings.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Fontanellaz, Maria Eugenia

Contributors dc:contributor

• Fischer, Rainer

• info:eu-repo/classification/ddc/570
• Phloëm
• Promotor <Genetik>
• Biowissenschaften, Biologie
• Forisomes
• Vicia faba
• Metaphloem development
• sieve element
• promoter

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-124105%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:62541