The Wall-Associated Kinase Gene Oswak11 Promoter In Oryza Sativa Responding To Abiotic Stress And The Preliminary Determination Of Its Kinase Domain Activity

The cell wall-associated kinase (WAK) family, which represents a unique receptor-like kinase (RLK) subfamily in plants, consist of an extracellular domain with similarity to the vertebrate epidermal growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic serine (Ser)/threonine (Thr) protein kinase domain. The extracellular domain which is covalently associated with cell wall through pectin can sense changes from the cell wall, while intracellular kinase domain can pass way the signal via phosphorylating. This construction make WAKs excellent candidates signaling molecules that directly link and allow communication between the cell wall and cytoplasm. The WAKs sequence were found in various plants. Scientists found that the WAKs expression can be induced by specific factors in plants. Gene regulation in higher plants is mainly carried out at the transcriptional level. The promoter sequence upstream of the plant gene contains a variety of cis-acting elements gene is critical to study specific features of gene expression and transcriptional regulatory mechanisms.A promoter sequence of length 974bp (-946/+28) upstream of the OsWAKll gene was successfully isolated form rice genomic by PCR technique. The promoter sequence was named as OsWAKpro. An analysis of the WAK promoter using the software databases revealed several czs-acting elements that may respond to environmental signals including TATA-box, CAAT-box, I-box, a W-box, as well as copper response elements (CuRE), methyl jasmonate (MeJA) regulatory elements, heat shock protein regulatory elements (HSE) and ABA response elements (ABRE).A plant expression vector (pBI121-OsWAKpro-GUS) including OsWAKpro and GUS reporter gene was constructed to study the regulatory mechanisms of the WAK promoter, and GUS activity was histochemically detected using the substrate X-gluc. Strong GUS staining was observed in the hypocotyls, cotyledons, first leaf, and petiole of cotyledons in transgenic OsWAKpro-GUS seedlings, and histochemical localization of GUS activity also revealed that the leaves and stems of OsWAKpro-GUS transgenic seedlings showed strong GUS activity in the xylem and cambium, but not in the root. The OsWAKI1 promoter was strongly upregulated in response wounding.To assess heavy metal-inducible promoter activity in stably transformed plants, OsWAKll expression in response to excessive metal stress in transgenic tobacco plants was examined using a β-glucuronidase (GUS) gene fusion. We observed significant levels of GUS activity in Al3+-, Na+-, and Cu2+-treated seedlings compared with control (35S-GUS transgenic) seedlings grown in the absence of metals. After treatment with excess metal ions for 24 h, the highest inducible GUS activity was observed in transgenic OsWAKpro-GUS seedlings treated with Cu2+(6.4-fold induction), followed by Al3+ (3.0-fold induction) and Na+(2.1-fold induction) compared with the control seedlings (without heavy metal stress). Cu also increased the level of GUS activity in the 35S-GUS transgenic seedlings. To acquire more detailed information on the effects of heavy metals on GUS, the effects of increasing Al3+, Na+, or Cu2+concentrations on GUS activity with MUG as the substrate was assayed using a Tecan Infinite M 200 Multifunctional Microplate Reader. GUS activity increased as a function of Al3+concentration from 0 (control, untreated with metals) to 100 μmol·L-1 and decreased thereafter. Seedlings were treated with Cu2+at the optimal concentration (10μmol·L-1) and Na+at the optimal concentration (50 mmol·L"-) for 24 h. We-found that the OsWAKpro responded to various metals, but its activity varied based on the metal species and concentration.An Omnia(?) Ser/Thr-Peptide Kit was used to test the kinase activity of the purified OsWAK protein in vitro. The kinase activity detected was produced in a reaction system containing 87.5 μmol·L-1 protein, the reaction system (concluding 1.0 mmol·L-1 ATP,0.2 mmol-L"1 DTT,10μmol·L-1 Ser/Thr-Peptide, at 30℃, and the 10μM peptide) was phosphorylate to saturation in 24 minutes. The analysis of OsWAK Michaelis constant (Km) and the maximum reaction rate (Vm) showed WAK performed kinase activity in vitro, and Km= 3.58μmol·L-1, Vmax=0.30 μmol·min-1 μg-1.