Cloning of two cysteine proteinase genes, CysP1 and CysP2, from soybean cotyledons by cDNA representational difference analysis

• Published in: Biochimica et biophysica acta 2003-06, Vol.1627 (2), p.129-139
• Main Authors: Ling, Jian-Qun, Kojima, Toshio, Shiraiwa, Masakazu, Takahara, Hidenari
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Base Sequence; Chlorophyll - chemistry; Cloning, Molecular; Cotyledon; Cotyledon - enzymology; Cotyledon - genetics; Cotyledon - growth & development; Cysteine Endopeptidases - genetics; Cysteine Endopeptidases - metabolism; Cysteine proteinase; DNA, Complementary - isolation & purification; Exons; Gene Expression Regulation, Plant; Genetic Techniques; Glycine max (L.) Merr; Glycine max - enzymology; Glycine max - genetics; Introns; KDEL motif; Molecular Sequence Data; Seeds - chemistry; Seeds - growth & development; Senescence; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Soybean Proteins - chemistry; Soybean Proteins - genetics; Soybean Proteins - metabolism
• ISSN: 0167-4781; 0006-3002; 1879-2634
• DOI: 10.1016/S0167-4781(03)00082-4

By cDNA representational difference analysis (cDNA RDA) and rapid amplification of cDNA ends (RACE), we isolated two cDNAs, CysP1 and CysP2, from the cotyledons of growing soybean ( Glycine max (L.) Merr.) seedlings. CysP1 cDNA is 1265 bp in size with a 1089-bp open reading frame (ORF), and CysP2 cDNA is 1270 bp in size with a 1089-bp ORF. Either CysP1 or CysP2 encodes a cysteine proteinase (CPR) with a C-terminal KDEL motif. The similarities between CysP1 and CysP2 are 93.5% in nucleotide sequences and 93.6% in deduced amino acid sequences. Furthermore, we determined the nucleotide sequences of CysP1 genomic DNA (1846 bp) and CysP2 genomic DNA (1831 bp). Both consisted of four exons and three introns. RNA-blot analysis revealed that both CysP1 and CysP2 were expressed from 6 days after germination (DAG) to 13 or 14 DAG in the cotyledons of growing seedlings and did so in a short period (9–12 DAG) in rejuvenated cotyledons. The transcripts of CysP1 and CysP2 were also detected in the root, flower and pod of soybean plants. Their physiological roles in the cotyledons of growing seedlings are discussed.