Idnettfic Ation And Analysis Of Wheat Vacuolar H~+-translocating Pyrophosphatase And Late Embryogenesis Abundent Protein Genes

When drought stress occurs, plants sense stress signal firstly by the receptors present on cell membranes, and then transfer the signal through intracellular signal substances, which results in expression regulation of drought-tolerance related genes. Products encoded by drought-induced genes protect plant cells directly or transfer the stress signal and regulate the expression of other drought responsive genes. Vacuolar H+-translocating pyrophosphatases (V-PPases) and late embryogenesis abundant proteins (LEAs) are not only necessary for plants growth and development, but also closely related with plants drought responses.In this work, three wheat V-PPase paralogs TaVP1, TaVP2and TaVP3were identified. Phylogenetic studies suggested that monocot grasses have three corresponding VP paralogs, as represented by the three wheat V-PPase genes. Most dicot plants have two VP paralogs, sharing the V-PPase gene ancestors with monocots. VP1and VP2paralogs are much more closer to each other than to VP3. The deduced polypeptide sequences of V-PPase genes are conserved in topological structure and most essential residues. Some amino acid substitutions between paralogs were known to have effects on their functions. TaVP3transcripts were only detected in developing seeds and no TaVP2transcripts were found in germinating seeds. TaVP2was mainly expressed in shoot tissues and was down regulated in leaves under dehydration. However, its expression was up regulated in roots under high salinity. TaVP1was relatively more ubiquitously and evenly expressed than TaVP2. Its expression level in roots was highest among the tissues examined, and was inducible by salinity stress.LEA genes have been identified through wheat EST database mining. Including53LEA genes reported before, a total of145contigs and15singletons of LEA genes have been identified. These contigs and singletons can be classified into eight groups according to the homology of their coding products to known LEA proteins. The numbers of contigs and singletons in each of the eight groups are much different. Contigs and singletons in each of the eight groups can be further classified, based on the principle of90%CDS nucleotide homology, into5,26,14,10,2,6,2and4subgroups, respectively. Contigs and singletons in Group2encode proteins called dehydrins. We found that wheat has at least seven types dehydrins different with structure domains. The two types genes coding YSK2and Kn types dehydrins account for the most of the members. Comparing with that of rice, obvious amplification of Group1-4LEA genes was found in wheat. The in silico Northern blot analysis showed that wheat LEA genes mainly expressed in reproductive tissues, roots, shoot tissues, seed tissues, and drought and cold stressed shoot tissues. Group1,5and6LEA genes mainly expressed in seeds. Group2and8LEA genes showed preferential expression in anthers or induced expression in dehydrated or cold stressed tissues. And some of Group2LEA genes also expressed in seeds. Some of Group3,4and7LEA genes mainly expressed in seeds, and others showed induced expression under dehydration and cold stress. The analysis of semi-quantitative RT-PCR of dehydrin genes supported the results of in silico Northern blot. Most dehydrin genes showed induced expression in dehydrated leaves, but only two subgroups showed induced expression in dehydrated roots. The expression of most dehydrin genes was induced by ABA treatment. The expression of three types dehydrin genes, i.e. Kn, KS and SK3, was induced by cold in both leaves and roots. The expression of Y2SK2, SK3, and some of YSK2and Kn type dehydrin genes was induced to different levels in salt stressed roots, but only the expression of SK3type dehydrin genes was induced in salt stressed seedling leaves.