| MicroRNAs (miRNAs), a type of RNA molecules that are generally19~22nt long,can interact distinct encoding genes by combination each other at their reversecomplimentary zone. Update, quantity of studies have confirmed that plant miRNAs playan important role in regulating the growth, development of eukaryotes and in mediatingthem to respond to diverse environmental factors by regulating the target genes at levelsof post-transcription and translation. In this study,32wheat miRNAs (TaMIRs) releasedin international miRBase database are selected to subject to investigation of theirexpression patterns under various Pi-supply conditions on which nine TaMIRs that areresonses to the low-Pi stress were identified. Meanwhile, the interacted target genes ofthe low-Pi responding TaMIRs were predicted based on a bioinformatic algorithm.Furthermore, the pri-sequence of TaMIR167that is response to low-Pi was cloned andsubjected to construction of the binary plasmids with modes of sense expression andanti-sense expression. The transgenic tobacco plants fused the anti-sense sequence ofTaMIR167were generated and further subjected to analysis of the role in mediating theplant tolerances to the low-Pi stress. The main results are as follows.1. Among the32tested wheat TaMIRs, nine numbers including TaMIR159b,TaMIR167, TaMIR399, TaMIR408, TaMIR1122, TaMIR1125, TaMIR1135,TaMIR1136and TaMIR1139were identified to be low-Pi responses. Of them,TaMIR408is down-regulated by the low-Pi stress as well as other eight numbers areup-regulated by the low-Pi stress.2. Using a bioinformatic technique, the putatively target genes that are interacted bythe nine low-Pi stress responding TaMIRs were predicted. The target genes could beclassified into various biological functions, such as those involved in cell cycling,chromosome rebuilding, signal transduction, primary metabolic pathway, phytohormoneresponse, transportation, defense responses and protein degradation, in addition thoseinvolved gene transcription regulation.3. Part target genes interacted by the low-Pi stress responding TaMIRs weresubjected to expression pattern analysis under various Pi-supply conditions. It was revealed that their expression levels under various Pi-supply conditions were opposite tothe TaMIRs. Theses results suggest that the low-Pi stress responding TaMIRs playimportant roles in mediating plants to respond and resist the low-Pi stress through theregulation of their interacted target genes at the post-transcriptional level.4. Using semi-quantitative RT-PCR, the pri-TaMIR167sequence, a low-Pi stressresponding wheat miRNA, was cloned. The binary plasmids fused the sense andanti-sense sequence of TaMIR167were constructed using DNA recombinant approach.Transgenic tobacco plants fused the anti-sense sequence of TaMIR167were generated.5. Under normal growth condition, non significant differences on plant phenotype,plant dry weight, total P content, and accumulative P amount per plant were observedbetween the wild type (control, CK) and seven transgenic lines with anti-senseexpression of TaMIR167.6. Under low-Pi stress, compared with the wild type, most of the transgenic tobaccolines among the seven tested ones displayed significantly improved ability to the low-Pistress. The transgenic lines with anti-sense expression of TaMIR167behaved muchlarger plant phenotypes, more dry mass and accumulative P per plant. However, the totalP contents between the wild type and the transgenic plants were similar each other.Taken together, our results suggest that TaMIR167, a low-Pi responding miRNA inwheat, has a potential role in generating germplasms of wheat and other crops withhigh-P usage efficiency. |
