Engineering for Expression of the Cold Regulated Barley Protein HVCR21 in E. coli

HVCR21 is a barley protein known to be cold regulated at the mRNA level. However, its function as well as the affect of low temperature on its translation are imknown. The purpose of this project was to engineer E. coli to express recombinant HVCR21. PGR primers were designed for the 5' and 3' ends of the HVCR21 coding region. The primers were also designed with a 5' Sad restriction site and a 3' Pst I restriction site. After ligation of the PGR product into the pGR4-T0P0 vector, bacteria were transformed and plated and the successfiil transformant verified by PGR. The new pGR4-TOPO-HVGR21 construct was therefore available as a ready source of the Sacl/Pst I fragment. The expression vector pQElOO was prepared by digestion with Sad and Pstl. The Sacl/Pst I fragment from pGR4-TOPO-HVGR21 was ligated to the SacI/PstI pQElOO vector. Transformants were analyzed by PGR with the HVGR21 primers. Sequence analysis of the engineered region of pQE100-HVGR21 confirms the correct orientation and frame for expression of HVGR21 in E. coli. Future studies will involve growth of bacteria containing this construct and SDS-polyacrylamide gel electrophoresis analysis of the expressed proteins. INTRODUCTION Low nonfreezing temperatures are required to acclimate plants to varying degrees of freezing tolerance (Levitt, 1980). Since the ability to cold acclimate is under genetic control, investigations have focused on the identification of cold-regulated genes (Thomashow, 1999). Several investigators have concentrated their efforts on characterizing barley and wheat cold-regulated genes. Ndong et al (2002) have described cold-regulated chloroplast proteins. Tsuda et al (2000), Vaguijfalvi et al (2000) and Gana et al (1997) are only a few of the investigators studying wheat cold-regulated genes. Brown (2001), Shen (2001) and Zhu (2000) have reported on stress-regulated barley multi-gene families. We isolated a number of full-length barley cDNA clones for cold-regulated genes (Ghang, 1993). Southern blot hybridization analyses revealed that two of these genes designated hvcrS and hvcrll belong to a small gene family (Gana et al., 1997). The transcript size for hvcr21 is 690 nt. Northern blot hybridization analyses revealed that BARLEY PROTEIN HVCR21 hvcrll is regulated by low temperature (Chang et al 1993). The function of the class of proteins encoded by this multi-gene family has yet to be elucidated. This study was undertaken to generate the necessary tools to examine the location and possible function of the encoded protein HVCR21 in planta. We have placed the cDNA insert corresponding to the complete coding region under the control of the lac promoter. Successful expression of the protein in E.coli will allow us to test an antibody generated against the imtmogenic region of the deduced amino acid sequence. The antibody will then be available for in planta studies. MATERIALS & METHODS Primer design The sequence of the coding region for pHVCR21 (Accession No, L28092, Chang et al, 1996) was analyzed and primers with Pst I and Sac I restriction sites designed and synthesized (Figure 1.). Two different restriction sites were chosen, since the aim was to directionally subclone the fragment. The 5' primer was designated primer 1. The 3' primer was designated primer 2. Isolation of HVCR21 ORF from pHVCR21 PCR amplification of HVCR21 ORF was performed with primers 1 and 2. Template pHVCR21(50 ng), 500 uM dNTP, primer 1 and primer2,5U Taq polymerase in a final volume of 50ul. Amplification was performed in thin walled reaction tubes in an Idaho Technology Rapid Cycler using the following program; Link (H2-C5-H4)= 94 C 50 sec, 95 C 30sec, 40 C 1 min, 72 C Imin, 29 cycles , 72 C 10 min. Subcloning into pCR4-TOPO The resulting amplicon was added directly to the ligation reaction containing the pretailed pCR4-T0P0 vector (Invitrogen life Technologies) and incubation performed at room temperature for 5 min[utes] then transferred to ice. One Shot TOPOlO cells (E. coli strain Topo 10 F' {laclq.}) were transformed with 2 ul of the PCR reactions. The transformation mixture (50 ul) was spread on LB 50ug/ml Kanamycin and incubated overnight at 37C. Verification that transformants contained first construct, pTOPO-HVCR21 A PCR master mix consisting of PCR buffer, dNTPs, primers land 2 and Taq polymerase at concentrationslisted above was prepared.Ten colonies were picked and individuallyspotted on an LB 50ug/ml kanamycin plate before resuspendingin 20 ul of the master mix. The reaction was placed in the Idaho Technology Rapid Cycler using the previouslydescribedprogramwith an additionalfirst 10 min[ute]94 C incubation. The inoculated plate was incubated overnight at 37 C and served as a sotu-ce for the positive