Identification Of Differential Expressed Genes Induced By Low-Pi Stress In Wheat Based On CDNA-AFLP Approach

As one of the indispensible inorganic nutrients, phosphorus is the important components of macro molecules in lives, such as nucleic acids, pospholipids, and ATP. In China, over half of the arable soils are in shortage of the available phosphorus, causing one of the factors for limiting the crop growth, yield formation and quality. Therefore, it has a vital role to promote the sustainable development of agriculture in China by improving the phosphorus acquisition and utilization efficiency in wheat (Triticun aestivum L.).So far, few studies on identification of the global differential expressed genes that responding to low-Pi stress have been reported in wheat. In this study, the genes differentially responding to various Pi supply and the treatment time of low-Pi in Shixin 828, a wheat cultivar with high-P use efficiency, have been explored, based on cDNA-AFLP approach and the bioinformatics tools. The main results were as follows:1. Using liquid culture method, the seedlings derived from normal Pi supply condition (2 mM Pi) and low-Pi (20μM Pi) treatment points of 1 h, 3 h, 6 h, 12 h, 24 h, 48 h, 72 h, and 96 h of Shixin828 were cultivated. The total RNAs from the roots of above samples were extracted with high quality.2. With a series of selected-primer pairs, in total 240, the transcripts of roots grown under normal Pi supply condition and various low-Pi treatment time points were identified based cDNA-AFLP approach. Among the about 2500 transcript derived fragments (TDFs) that effectively PCR amplified with the selected-primer pairs, 70 of TDFs with repeated occurrence were detected. By further re-PCR amplification, cloning, and sequencing, in total 52 TDFs with mostly of base lengths in 100-450 bp were figured out.3. Among the 52 differential expressed genes responding to low-Pi stress, 6 could not be given homologous sequences and 22 were unknown biological functions though with known homologous sequences in GenBank. 24 with known homologous sequences had putative biological functions.4. The 24 TDFs with known putative functions could be classified into the groups of signal transduction, transcriptional regulation, metabolism, development, and stress response. The TDF (TDF83-1) involved in signal transduction was Ndr(nuclear Dbf2-related)protein, a member of Ser/Threo kinase subfamily. The TDFs classified into transcriptional regulation included four transcription factors, such as 2 zinc-finger proteins (TDF47-1and TDF106-1), 1 bHLH type protein (TDF106-2), and 1 bZIP type protein (TDF66-3). 1 RNA-binding protein (TDF59-3), and 1 polyadenylation specificity factor (TDF98-1). The TDFs grouped in metabolism included GDP dissociation inhibitor alpha (TDF5-1), pyruvate kinase (TDF66-1), DNA polymerase V family protein (TDF59-2), nuclear distribution gene C (TDF80-2), CENP-E like kinetochore protein (TDF102-1), acyl-CoA oxidase (TDF95-2), ubiquinol-cytochrome-c reductase (TDF94-1), and cyclase-associated protein (TDF43-1). The TDFs in development class had 1 alpha-expansin OsEXPA24 (TDF95-1) and 2 cellulose synthase catalytic subunits (TDF102-2 and TDF81-2). The TDFs responding to stress stimuli included 2 salt-responsive (TDF79-2 and TDF80-3), cold-responsive (TDF68-1), abiotic inducible (TDF82-1), powdery mildow infected inducible (5-2), and fusarium graminearum infected inducible (TDF81-1)。5. It is suggested that the response to low-Pi stress on physiology, biochemistry and phenotype in wheat is a comprehensive results at the molecular level, in which the distinct signal transduction and transcriptional regulation play key roles by their further regulation of the downstream genes involved in cellular metabolism, development, and response or defense. In this study, the differential expressed genes with various functions should be functional on responding to low-Pi stress and improving the phosphorus use efficiency under deficient-Pi condition in Shixin828.