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Molecular cloning of yieldins regulating the yield threshold of cowpea cell walls: cDNA cloning and characterization of recombinant yieldin
Author(s) -
OkamotoNakazato A.,
Takahashi K.,
Kido N.,
Owaribe K.,
Katou K.
Publication year - 2000
Publication title -
plant, cell and environment
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.646
H-Index - 200
eISSN - 1365-3040
pISSN - 0140-7791
DOI - 10.1046/j.1365-3040.2000.00526.x
Subject(s) - complementary dna , recombinant dna , microbiology and biotechnology , biology , molecular cloning , amino acid , vigna , signal peptide , nucleic acid sequence , peptide sequence , escherichia coli , biochemistry , gene , botany
cDNA for yieldin of Vigna unguiculata L. was cloned with reverse transcriptase‐polymerase chain reaction using synthetic oligonucleotides as primers. The primers were designed on the basis of the N‐terminal amino acid sequence of the yieldins isolated from the wall preparation of cowpea hypocotyls. The 1·2 kbp cDNA for yieldin contained an open reading frame of 981 base pairs, encoding 327 amino acids including 23 amino acids as a putative signal sequence. An homology search of the deduced amino acid sequence revealed that the yieldin was homologous to acidic class III endochitinases (EC 3·2.1·14) and concanavalin B. A cDNA fragment containing the yieldin‐coding region was introduced to Escherichia coli cells using an expression vector to express the recombinant protein. The recombinant yieldin was obtained from the recombinant E. coli and its effect on the wall mechanical properties was examined by reconstitution experiments. The recombinant yieldin fully restored the acid‐induced change of the yield threshold tension ( y ) of heat denatured glycerinated hollow cylinders (GHCs) of cowpea hypocotyls. Northern hybridization analysis revealed that the yieldin mRNA was expressed mainly in the rapid and moderate elongation region of the etiolated hypocotyl of Vigna unguiculata L.