Studies On The Differential Expression Of CDNAs Induced By Water Stress In Wheat Seedling Stage

Drought is one of the most important environmental factors that cause osmotic stress and dramatically limit plant growth and crop productivity. Therefore, it is urgent to study the drought- resistant mechanism and breed new drought-resistant wheat varieties to feed the world's growing population.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 Peng and Dr. Pardee. The technique is simple, time-saving, sensitive and reproducible.In present research, wheat seedling stressed in 16% PEG-6000 (polyethylene glycol, MW=6000, -0.5MPa) solution was used as treatment sample, and wheat seedling cultured in distilled water as control sample. mRNA differential display and silver staining techniques were adopted to isolate cDNA in wheat seedling. A total of 52 differential expression cDNA fragments were obtained. 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 and visualized by silver staining. The length range of differential displayed PCR products mostly extended from 150 bases to 750 bases. Among the total 52 cDNA fragments , only 15 positive cDNA fragments have been cloned and sequenced after being testified by Reverse Northern. Four were up-regulated and eleven down-regulated cDNA fragments . When amplified by single arbitrary primer, all of the above fragments appeared negative. These fragments were cloned in E.coli DH10B competent cell after having been ligated with the pGEM?T Easy Vector . By sequencing and then querying EST database of GenBank, we canfind that eleven of them have high homologous sequences while the other 4 were poor. Of these eleven fragments, WSI732 was 86% homologous to a pathogen induced Sorghum bicolor cDNA c\one.WSI483 was 100% homologous to a cDNA clone of heat-stressed wheat flag leaf. WS1153 was 100% homologous to a cDNA clone of wheat 20 days spike. Eight fragments were highly homologous to other cDNAs in wheat, rice, barley, broomcorn, respectively. The others four were low homologous (less than 30%) to EST library and were supposed novel genes, whose functions need be further studied. Especially, WSI699 strongly expressed after being stessed 48 hours later. Reverse Northern proved the same result. Aligning with Genbank database, we find that there was no homologous sequences to it. Therefore, it most probably be a novel gene, and there is still much work to do for its full length and its function identification.