Isolation and characterization of wheat triticin cDNA revealing a unique lysine-rich repetitive domain.
Polyclonal antibodies were raised against a purified 22 kDa triticin polypeptide (delta) and were used to screen a wheat seed cDNA library in the Escherichia coli expression vector lambda gt11. The isolated cDNA clones were grouped into three families based on their cross-hybridization reactions in DNA dot-blot studies. Southern blots of genomic DNAs extracted from ditelocentric and nullisomic-tetrasomic lines of Chinese Spring wheat, probes with the excised cDNA inserts, indicated that one of the three families (9 clones) had triticin clones. This was finally confirmed by comparing the predicted amino acid sequences of two of these clones (lambda Tri-12, lambda Tri-25) with the published tryptic peptide sequences of triticin. The Southern blots also showed that there is at least one triticin gene located on the short arm of each of the homoeologous group 1 chromosomes (1A, 1B, 1D), although till now no triticin protein product has been identified for the chromosome 1B. The nucleotide sequence of the largest triticin cDNA clone lambda Tri-25 (1567 bp) is presented here, and its predicted amino acid sequence shows strong homology with the legumin-like proteins of oats (12S globulin), rice (glutelin) and legume seeds. A unique feature of the triticin sequence is that it contains a lysine-rich repetitive domain, inserted in the hypervariable region of the typical legumin-like genes. Northern blotting of total RNA extracted from different stages of the developing wheat seed revealed that the triticin gene expression is switched on 5-10 days after anthesis (DAA). There was a steady increase in the level of triticin mRNA until 20 DAA, after which it started decreasing. The maximum mRNA accumulation occurred between 17 and 20 DAA. These observations conform closely with the published data on triticin protein accumulation during grain development.
Centre for Cereal Biotechnology, Waite Agricultural Research Institute, Glen Osmond, Australia.
N K
NK
Singh
G R
GR
Donovan
H C
HC
Carpenter
J H
JH
Skerritt
P
P
Langridge
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017343:Genes, Plant / Q000235:genetics; D008969:Molecular Sequence Data; D010940:Plant Proteins; D014674:Plant Proteins, Dietary / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D012639:Seeds / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000196:embryology / Q000235:genetics*; D000097793:Legumins
D010940:Plant Proteins; D014674:Plant Proteins, Dietary; D012333:RNA, Messenger; D000097793:Legumins