Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene

Dhundy R. Bastola, Vaijaynti V. Pethe, Ilga Winicov

Research output: Contribution to journalArticle

63 Citations (Scopus)

Abstract

Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation. We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner. The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences. The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding. DNA binding of the recombinant Alfin1 was inhibited by EDTA. Alfin1 mRNA was found predominantly in alfalfa roots. Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels. Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection. Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants.

Original languageEnglish (US)
Pages (from-to)1123-1135
Number of pages13
JournalPlant Molecular Biology
Volume38
Issue number6
DOIs
StatePublished - Dec 1 1998

Fingerprint

alfalfa protein
Medicago sativa
zinc finger motif
Zinc Fingers
Zinc
alfalfa
Salts
Genes
promoter regions
salts
oligonucleotides
Proteins
genes
Oligonucleotides
Messenger RNA
clones
DNA
Salt-Tolerant Plants
Complementary DNA
Clone Cells

Keywords

  • Alfalfa
  • DNA binding
  • Roots
  • Salt tolerance
  • Zinc finger protein

ASJC Scopus subject areas

  • Agronomy and Crop Science
  • Genetics
  • Plant Science

Cite this

Alfin1, a novel zinc-finger protein in alfalfa roots that binds to promoter elements in the salt-inducible MsPRP2 gene. / Bastola, Dhundy R.; Pethe, Vaijaynti V.; Winicov, Ilga.

In: Plant Molecular Biology, Vol. 38, No. 6, 01.12.1998, p. 1123-1135.

Research output: Contribution to journalArticle

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AB - Alfin1 cDNA, obtained by differential screening of a poly(A)+ library from salt-tolerant alfalfa cells, encodes a novel protein with a Cys4 and His/Cys3 putative zinc-binding domain that suggests a possible role for this protein in transcriptional regulation. We have expressed the cDNA in Escherichia coli and show that the recombinant Alfin1 protein binds DNA in a sequence-specific manner. The DNA recognition sequence was determined from individual clones isolated after four rounds of random oligonucleotide selection in gel retardation assays, coupled with PCR amplification of the selected sequences. The consensus binding site for Alfin1 is shown to contain two to five G-rich triplets with the conserved core of GNGGTG or GTGGNG in clones showing high-efficiency binding. DNA binding of the recombinant Alfin1 was inhibited by EDTA. Alfin1 mRNA was found predominantly in alfalfa roots. Growth of salt-sensitive Medicago sativa L on 171 mM NaCl led to a slight decrease in Alfin1 mRNA, while the salt-tolerant plants showed no decrease in Alfin1 mRNA levels. Interestingly, recombinant Alfin1 binds efficiently to three fragments of the MsPRP2 promoter, each containing consensus sequences identified by the random oligonucleotide selection. Since MsPRP2 transcripts were shown to be root-specific and accumulated in alfalfa roots in a salt-inducible manner, Alfin1 may play a role in the regulated expression of MsPRP2 in alfalfa roots and contribute to salt tolerance in these plants.

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