Cloning and sequencing of the cDNA for cas17, a cold acclimation-specific gene of alfalfa.

We have previousb reported On two cold acclimationspecific (cas) genes from alfalfa, casl8, coding for a dehydrinrelated protein (Wolfraim et al., 1993), and casZ5, coding for a putative nucleus-targeted protein (Monroy et al., 1993). We report here the cloning and sequencing of a new cDNA clone, pAcs784A, corresponding to a gene, casl7 (cas gene coding for a 17-kD protein), that is induced by cold. Twoweek-old seedlings of a freezing-tolerant cultivar of alfalfa (Medicago sativa L. ssp. falcata cv Anik) were cold acclimated at 5OC for 1 week. A X Uni-Zap cDNA library was constructed using poly(A)' RNA isolated from these cold-acclimated seedlings. The library was screened by in situ plaque hybridization using a partial-length cDNA clone (pSM784) that has been previously shown to correspond to a cold-inducible gene family (Mohapatra et al., 1989). The cDNA clone described here, pAcs784A, represents the gene casl7, which shows extensive homology with casld (Wolfraim et al., 1993) and belongs to the same gene family. The characteristic features of the cDNA clone pAcs784A are described in Table I. It is 809 bp long, contains an open reading frame of 480 bp, and includes a putative polyadenylation signal. It can encode a polypeptide (CAS17) of 160 amino acid residues with a predicted molecular mass of 16.7 kD. CAS17 is rich in Gly and Thr, which together constitute 42 mo1 % of the protein. Of the 160 amino acid residues of the CAS17 sequence, 102 (64%) match those of CASl8 reported earlier (Wolfraim et al., 1993). CAS17 also shows high sequence homology to members of the Group 2 LEA/ dehydrin/RAB family of proteins. Two similar motifs common to CASl8 and members of the Group 2 LEA/dehydrin/ RAB proteins also occur in CAS17: DKIKEKIPG and GEKKGIMEKIKEKIPG. As was the case with CASl8 (Wolfraim et al., 1993), there are regions of extensive (Gly-Thr), repeats, where 1~ is Feater than 2, One such repeat consists of 10 consecutive Gly-Thr pairs. Such (Gly-Thr), repeats are characteristic of proteins of other organisms (Li-Weber et 1987). circadian regulation of plant ,-hil]ing resistance has been demonstrated (Rikin, 1991), and low temperature has been shown to inter-