| Drought is one of the most important environmental factors that cause osmotic stress and dramatically limit plant growth and crop productivity. It's an important branch of plant research on stress resistance to enhance or improve the capability of drought and water deficit—tolerance in plant. With the advance of molecular biological technique, bioengineering becomes a rapid efficient method of improving plant resistance to drought, water deficit, on the viewpoint of many researchers.Differential display reverse transcription polymerase chain reaction (DDRT-PCR), one of the most widely employed techniques to identify differential gene expression, has become a powerful tool in the area of molecular genetics since 1992 when the technique was invented by Liang and Pardee. The technique is simple, time-saving, sensitive and reproducible.In present research, we adopted both mRNA differential display and silver staining techniques to isolate grain sorghum cDNA induced by 19.5%(-0.7MPa)PEG-6000, total 53 differential expression cDNA fragments were obtained. Sorghum seedling in 19.5% PEG-6000 (polyethylene glycol, MW=6000, V= -0.7MPa) solution was used as treatment sample, and sorghum seedling in dH2O as control sample. Differential expression of mRNA between treatment sample and control sample was analysed by DDRT-PCR. The PCR amplification products were separated by 6% denaturing polyacrylamide gels. Distinct bands were visualized by silver staining. A total of 53 differential expression cDNA fragments had been detected, including 6 absolute expression fragments induced by water stress, 42 enhanced expression fragments, 5 inhibited expression fragments. After further certified with reverse northern dot blot, thirteen fragments are proved to be positive. When amplified by single arbitrary primer, all of the above fragments appeared negative. These fragments were cloned in the pGEM?-T Easy Vector . Through sequencing and then querying EST database of GenBank, we can find that ten of them have high homologous sequences while the other 3 have poor. Of these ten fragments, T11A—AP3 was 100% homologous to wounded leaves Sorghum bicolor cDNA clone . Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. Acid- and alkaline-treated roots Sorghum bicolor cDNA clone and Salicylic acid-treated seedlings Sorghum bicolor cDNA clone. T11G—AP3-2 was 100% homologous to Oxidatively-stressed leaves and roots Sorghum bicolor cDNA clone. T11G—AP4 was 100% homologous to Abscisic acid-treated seedlings Sorghum bicolor cDNA clone. Iron-deficient seedlings Sorghum bicolor cDNA clone and Salt-stressed seedlings Sorghum bicolor cDNA clone. Other seven fragments were more than 95% homologous to the Sorghum bicolor cDNA clone induced by Abscisic acid-treated , wounded , Oxidatively-stressed , Acid- and alkaline-treated, Salt-stressed, Drought-stressed before flowering, Light Grown and Iron-deficient. The others there were low homologous to EST library and were supposed novel genes, whose functions need further study. Therefor, it most probably be a novel gene, and there is still much work to do for its full length and its function identification. |
