BioSample

Maize (Zea mays L.) inbred line DH4866 was used in this study. Plants grew in flowerpots containing field soil under natural conditions. When the tassel spread out of the uppermost leaf, some plants were treated with drought stress for 7 days, and others were left under normal-watered conditions as control. In the period of the stress, the middle part of the top fully expanded leaves of the plants under water stress treatment and the control were taken at 8:00 am each day, and the leaf osmotic potential was measured by the Fiske Micro-Osmometer (FISKE ASSOCIATES, USA). The stressed plants were watered every day to maintain a similar water-deficiency state by adjusting water supply, according to the leaf osmotic potential. And the leaves osmotic potential of the stressed plants was hold to -0.4 to -0.5Mpa, while the osmotic potential was -0.2 to -0.25Mpa in the leaves of the control plants. During the stress, there were visible signs of water deficiency such as leaf rolling during the daytime, though at night the rolled leaves spread out. Maize plants were stress-treated at the beginning of heading stage. After 7 days of water deficiency by withholding water, the plants began to enter the flowering stage. The maize flag leaves were harvested at 168h (7 day) of stress treatment, frozen in liquid nitrogen immediately and stored at -80oC for further analysis. Unstressed plants as controls were harvested at the same time as the stressed plants. And one sample consisted of the leaves from three independent plants under the same condition. Total RNA was isolated respectively from samples of stressed and control leaves by water-saturation phenol-guanidine isothiocyanate-chloroform method and mRNA was extracted from the RNA pool using an OligotexTM mRNA Purification Kit (QIAGEN, USA). Forward-SSH library was constructed using cDNA synthesized from treated plants as tester and that from the control plants as driver with the Clontech PCR-SelectTM cDNA subtraction Kit (CLONTECH, USA) according to the manufacturer's protocol.