In vitro production of selected C-repeat binding factors (CBF) encoded by group five chromosomes in winter wheat cv. Norstar

Abstract

Winter wheat has the potential to produce 20-30% higher yields than spring wheat if it successfully overwinters. Due to early establishment in the spring, winter wheat can more efficiently utilize spring moisture, out-compete weeds, mature early and usually circumvent Fusarium head blight infections. However, in some regions on the Prairies production of winter wheat is constrained due to insufficient winter-hardiness in current varieties. To develop winter wheat varieties with increased cold hardiness, it is essential to gain more insight into the molecular mechanisms underlying low temperature (LT) tolerance. Genetic analysis has revealed a major quantitative trait locus (QTL), FrA2 on chromosome 5A that explains 50% of LT tolerance in winter wheat cv. Norstar. Minor QTLs for LT tolerance have also been identified on chromosomes 5B and 5D. Physical characterization of group 5 QTLs has revealed the presence of a number (≥ 60) of C-repeat binding factors (CBF), which function as transcription activators of several cold regulated genes (COR). The CBFs bind to a GC-rich region, also known as the CRT motif, present in the promoter of COR genes. Hence, determining CBF functional characteristics and their affinity towards the CRT motif would help to understand the function of each CBF isoforms and its role in LT tolerance in winter wheat. In planta transcription factors are present in small quantities, but are needed in relatively large quantities for functional analyses. Therefore to study the functional characteristics of CBF, they must be produced in abundance, which can be achieved by producing these proteins in recombinant form. The main objective of this work was to optimize methods to produce wheat recombinant CBF in vitro in quantity and quality suitable for functional studies. Thirteen CBFs encoded by group 5 chromosomes of winter wheat were cloned from a previously constructed BAC (Bacterial Artificial Chromosome) library using DNA from a cold hardy winter wheat cv. Norstar. Twelve out of 13 CBF genes were inserted into protein expression vectors that were transformed into E. coli BLR(DE3)pLysS cells to produce TrxHisS–CBF fusion proteins. To extract recombinant CBF fusion proteins, E. coli cells over-expressing them were disrupted under high pressure in a cell homogenizer for 2-5 min to obtain the TrxHisS-CBFs in cell soluble extracts. Recombinant CBFs were purified from the cell soluble extract with affinity and ion exchange chromatography procedures using an automated fast protein liquid chromatography (FPLC) system. Adequate quantities of 15 recombinant CBFs of winter wheat cv. Norstar were purified and selected TrxHisS-CBFs from them were assessed for functional studies. Denaturing gel electrophoresis revealed that 11 out of 15 TrxHisS-CBFs migrated slower on SDS-PAGE which could be due to their shape, charge and presence of a large number of proline residues. Eight of the purified TrxHisS-CBFs were tested for their affinity towards the CRT motif by electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR). EMSA assay showed differences in in vitro DNA binding ability between the recombinant CBFs encoded by group 5 chromosomes. The SPR assay suggested that compared to TrxHisS-CBFIIId-A12 (FrA2), TrxHisS-CBFIIId-B12 (FrD2) had almost eight times more affinity towards the Wcs120 promoter CRT motif. In conclusion, a combination of growing E. coli cells at low temperature (28°C), induction at low A600nm, use of NPI-10 extraction buffer with mechanical disruption under high pressure by cell homogenizer resulted in all TrxHisS-CBF fusion proteins in cell soluble extracts. Affinity chromatography followed by ion exchange chromatography using a fast protein liquid chromatography (FPLC) based automated purification method proved to be optimal to purify recombinant TrxHisS-CBF fusion proteins of winter wheat cv. Norstar which could be suitably used for functional studies.