The Molecular Characterization, Expression Patterns Of Wheat CDPKs And Genetic Transformation Of TaCDPK1

Acting as the important conserved signal transduction components of abiotic stress cues, calcium-dependent protein kinase (CDPK) exist with multi-numbers in plants. So far, there were lots of reports, such as identification, characterization of CDPKs in Arabidopsis and rice, but few studies of CDPKs in wheat (Triticum aestivum L.) have been carried out. In this study, five wheat CDPK genes (TaCDPK1 to TaCDPK5) were isolated based on bioinformatics approach. The molecular characterizations, expression profiles under various-Pi conditions of the five wheat CDPK genes have been analyzed. In the meantime, the functions of TaCDPK1 on improving the low-Pi stress was evaluated based on the transgenic tobacco plants in which the plants was integrated the open reading frame (ORF) of TaCDPK1. The main results were as follows:1. In the international bioinformatics website NCBI, the wheat CDPK cDNA sequences released in the GenBank have been searched. In total five wheat CDPK genes of which characterized and functions are scarce have been isolated. In this study, they were designated as TaCDPK1 to TaCDPK5, respectively. The cDNA lengths of 5 CDPKs changed from 1654 bp (TaCDPK1) to 1960 bp (TaCDPK4), with the open reading frames (ORFs) from 1542 bp(TaCDPK5)to 1677 bp (TaCDPK3), and the translated amino acids of 513 (TaCDPK5) to 558-aa (TaCDPK3). TaCDPK1 to TaCPK5 contained the distinct conserved domains of plant CDPKs, including one protein kinase domain and four EF-hands calcium-binding domain. The phylogenetic tree analysis indicated that the five wheat CDPKs derived from different ancestors.2. Using Shixin828, a high-P use efficiency cultivar to be the material, the expression profiles of TaCDPK1 to TaCDPK5 were conducted. It is found that the expression levels of TaCDPK1 and TaCDPK5 were up-regulated by low-Pi stress. There were similar transcripts of TaCDPK2 and TaCDPK4 in roots and leaves, with a pattern of non-change responding to various-Pi supplies, suggesting these genes have a constitutive expression pattern. Under the sufficient and deficient-Pi conditions, there were no transcripts of TaCDPK3 detected. Taken together, TaCDPK1 and TaCDPK5, the genes responding to low-Pi stress, are possibly involved in the process of plants to transduce the low-Pi stress signal and the plant responding and resistant of Pi-starvation stress. 3. Using DNA recombinant technology, the binary cassette fused the open reading frame (ORF) of TaCDPK1 has been constructed. The transgenic tobacco plants ectopically expressed TaCDPK1 were generated by Agrobacterium-tumefaciens mediated transgenic method. Under deficient-Pi condition, the tobacco plants with high expression level of TaCDPK1 had obviously improved phosphorus acquisition capability, showing it having much more phosphorus accumulative amount, higher fresh weight per plant and dry weight per plant than those in CK (the tobacco plants transformed the empty binary vector pCAMBIA3301). The tobacco plants with high expression levels of TaCDPK1 also had higher photosynthetic pigment contents, soluble protein contents and soluble sugar contents than those in CK. Therefore, the tobacco plant with higher expression of TaCDPK1 had improved physiological parameters, growth and development under low-Pi condition was mainly resulted from its increased Pi acquisition capability, which further enhances the accumulative phosphorus amount of plants.