Characterization And Expression Analyses Of The H~+-pyrophosphatase Gene From Rye

The H+-pyrophosphatase (H+PPase) is a kind of enzyme which can hydrolyze PPi, and it exists in a range of species including plants, protozoans, algae and bacteria. It is believed that the expression of H+-PPase was induced by N, P, K deprivation and low temperature, drought, high salt, Cd’+, respectively. Thus characterization of H+PPase could be helpful for crops resistance to environment stress.In this study, we first explored the physiological conditions of wheat and its relatives under K deprivation. Then we cloned the full length cDNA sequence of ScHPl from rye by using RACE, and further characterized the gene structure, sequence, expression pattern of ScHP1. The main results are as follows:1. The materials with tolerance to potassium deficiency were identified for research object in the following study.2. The putative ScHP1cDNA sequence of rye was 2629 bp with an open reading frame (ORF) of 2289 bp encoding 762 amino acids. And the protein molecular mass was 79.4 kDa. The ScHPl gene sequence of rye was 4354bp.3. Structure prediction indicated that ScHP1 contained 14 transmembrane domains, and tertiary structure prediction indicated that the protein contained two chains, one potassium ion, and ten magnesium ion binding sites. Aliment of the sequence of wheat and other species found that the 5’terminal regions of the proteins varied greatly while the remaining parts of the sequences were highly conserved. The prediction of rye ScHP1 protein domains suggested that the primary structure was characterized by conserved domains of the H+-PPase protein family such as DVGADLVGKVE, EYYT, and GNTTAA.4. The full-length ORF was inserted into the expression vector pA7-YFP. The transient recombination expression vector of pA7-ScHP1-YFP (yellow fluorescent protein) was constructed to confirm the subcellular localization of ScHP1 in onion epidermal cells. Our results indicated that cells transformed with pA7-ScHP1-YFP showed a yellow color on plasma membrane, whereas cells transformed with the control vector (pA7-YFP) showed a widely distributed yellow color. These results suggest that the ScHP1 protein is localized to the plasma membrane.5. The responses of ScHP1 to N, P, and K deprivation, as well as cold, drought, and high salt stress, were investigated over 72 h in different tissues. In leaves under N deprivation, cold, and high salt stresses, the expression level of ScHPl increased gradually from the beginning to the end of the experimental period. However, under treatments of P and K deprivation and drought, the transcript abundance of ScHP1 in leaves gradually increased to a peak at approximately 36-48 h and then decreased to a lower level. In roots, ScHP1 expression levels were initially low, then gradually increased through the end of the experiment in response to all stresses except K deprivation, for which ScHPl expression peaked at 48 h and then decreased. In stems, under the P and K deprivation, cold, and high salt treatments, ScHP1 expression was gradually down-regulated as time progressed. The expression level of ScHP1 in stems was very low. These findings further demonstrated that ScHP1 play an important role in stress resistance.