ABA-Binding Protein Encoding Gene Cloning, Expression In Yeast And Specific-ABA Binding Of Its Product

Abscisic acid (ABA) acts as a plant hormone, not only regulates many important aspects of plant growth and development, including dormancy, senescence and fruit development, but also mediates some aspects of physiological responses to environmental stresses such as drought, cold and salt. After arrival at its site of action, the first step in ABA response is expected to be some kind of perception and recognition, ABA receptor plays a key role in the signal recognition. The use of ABA analogs in germination and gene expression bioassays has suggested the existence of multiple ABA receptors with different structural requirements for activity in different response pathways. The greatest progress has been made using genetic, biochemical and cell biological approaches to study ABA binding proteins, however, no ABA putative receptors have been identified to date. Consequently, amount of work have yet to be done to identify real ABA receptor.Recently an abscisic acid binding protein with high specificity and affinity for ABA was purified from epidermis of Vicia faba L. in our laboratory, and further characterized using various biochemical techniques, there is evidence to link the protein to the physiological effects of ABA in some aspects. The above-mentioned work may result in the molecular cloning the gene encoding ABA binding protein. In this research two full-length cDNAs have been cloned by a combination of RT-PCR and 3' -IS1 -RACE with synthesized degenerate primers from young leaves of Vicia faba L., Pichia methanolica high-level expression systems of the genes have been constructed, and the milligram expressed protein was purified using ProBond resin purification system, which may result in further identification of the function of the ABA binding protein.The full-length cDNA of ABP370 fragment is 3449 bp long and has an open reading frame of 2304 bp encoding 768 amino acids with 876 bp long 5'-UTR , 369 bp long 3'-UTR and Poly(A) tail. The full-length cDNA of ABP640 fragment is 1012 bp long and has an open reading frame of 780 bp encoding 260 amino acids with 88 bp long 5'-UTR, 144 bp long 3 '-UTR and Poly(A) tail.The coding sequence of the two full-length cDNAs were cloned by PCR, and inserted into expression vector pMET a between the downstream of a secreting signal peptide and the upstream of 6x histidine in the same reading frame with the coding sequence. The secreting recombinant expression vectors pMET a B/ABP2304 and pMET a A/ABP780 were constructed and transformed into PMAD16 with LiCI transformation. The Ade+ transformants were selected and fermented in flasks with 20ml BMMY medium, then, induced by 0.5% methanol. The expression protein was analyzed by SDS-PAGE after five days of induction. SDS-PAGE analysis revealed that the high-level expression recombinant strains of PMAD16/ pMET a B/ABP2304 and PMAD16/ pMET a A/ABP780 had specific bands at 75kD and 55kD separately, account for 30% and 10% of the total protein separately, which were purified using ProBond resin purification system, and obtained 15mg at levels above 0.75g/L and 7mg expression protein at levels above 0.35g/L separately once purification, the purity is both above 90%. The method of labeled-Iigand analysis revealed that the 75kD purified protein had high specificity and affinity for ABA, the 55kD purifiedprotein had no specific affinity for ABA.