Isolation Of Heat-stable Proteins In Maize Seedling Roots Under Osmotic Stress And Its Expression Regulation By Signal Substances

The Lu Dan No.50 variety in maize(Zea mays) was cultivated by solution and treated under osmotic stress with PEG and NaCl. A novel heat-stable protein was extracted, isolated, and its molecular weight was mensurated.The maize seedling roots were cultivated under isotonic nonionic(20%PEG)and ionic(0.82% NaCl)osmotic stress,simultaneously treated by different signal substances for 96h, and then the heat-stable proteins of the maize seedling roots under different treatments were detected by SDS--PAGE. At last quantitatively analyzed by stained-gels-scanning. The main results are as follows:The heat-stable protein was extracted. Fistly, the treated maize seedling roots were ground into a fine powder, and extracted for 20 min at 4℃by 1.5 volumes of extraction buffer (100mmol/L Tris-HCl, 4mmol/L EDTA-Na2, 1mmol/L PMSF, pH 7.0), then centrifuged at 10 000 ╳g for 20 min at 4℃. The supernate containing total soluble proteins was heated in water bath for 10 min at 80℃, centrifuged for 10 min at 6 000 ╳g ,4℃. Finally, the supernate containing heat-stable proteins was precipitated by ice acetone, then centrifuged, the pellet was resuspended in sample butter, after short centrifuged, the supernatant were detected by 15% SDS--PAGE.2. The novel heat-stable protein was discoved. In contrast to control ,maize seedling roots adapted to grow in medium containing different concentration of ABA, NaCl and PEG produced several enhanced or new polypeptide bands on SDS--PAGE. The 63.5KD polypeptide( refered to it as ZmR63.5)was presented in both different concentration of ABA and osmotic-induced roots, but was not detected in natural roots, and the proportion of its content in the total heat-stalbe proteins was large. After osmotic stress and rewater treatments,the results showed that: ZmR63.5 was related with dehydration,so it should be drought-induced protein.3.The concentrations of NaCl and PEG for signal substances were confirmed. After treated by different concentrations of PEG & NaCl for different time, the maize seedling roots were detected by SDS—PAGE. The results showed that: the content of ZmR63.5 treated by 20%PEG for 96h was better. So the best choice is that: isotonic(ψs =-0.66 MPa) nonionic(20%PEG)and ionic(0.82%NaCl) osmotic stress for 96h.4.The signaling pathway of ZmR63.5 protein in response to stress. Maize seedling roots were treated by isotonic nonionic and ionic osmotic stress,simultaneously fed with ABA, neomycin sulfate and Butyl alcohol (PLD/PLC inhibitor), EGTA (Ca2+ inhibitor), TFP (CaM inhibitor), K+, TEA (K+ channel inhibitor), and SA for 96h, respectively. The varying content of ZmR63.5 was detected by SDS--PAGE. The results indicated that: under different osmotic-stresses, the signal substances ABA, PLD/PLC, Ca2+ had the similar effect on the expression of osmotic-induced ZmR63.5 protein; while CaM, SA and K+ had different effect on it.