Exploring The Differentially Expressed CDNA In Germinating Wheat Induced By Osmotic Stress

Wheat is fundamental grain in the majority of countries. 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 shoot germinating in 16% PEG-6000 (polyethylene glycol, MW=6000, ?= -0.5MPa) solution was used as treatment sample, and wheat shoot germinating 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. The length range of differentially displayed PCR products extended from 200 bases to about 500 bases. Sixteen differential expressed cDNA fragments were obtained, including thirteen up-regulated and three down-regulated cDNA fragments. When amplified by single arbitrary primer, all of above fragments appeared negative. These fragments were cloned in the pUCm-T vector. Through sequencing and then querying the database of GenBank, two fragments, WSI241 (water-stressed induced) and WSI208, are highly homologous to the known genes, but the others are ESTs (expressed sequence tags) with unknow function. Among them, the homologous of WSI241 to Em gene is 100%, WSI208 to ARA-1 mRNA is 100%, WSI483 to drought-stressed wheat seedling cDNA is 100%, WSI301 to salt-stressed wheat leaf cDNA is 98%; WS267 to heat-stressed wheat flag leaf cDNA is 93%, WSI289 to Wheat ABA-treated embryo cDNA is 92%, WSI268 to Wheat Fusarium graminearum infected spike cDNA is 97%. After further certified with reverse northern dot blot, four fragments are proved to be positive, can be used as probe or selected primer to do further research.