The Screening And Function Analysis Of Arabidopsis Osmotic Stress Responsible Genes

Adverse environmental stress such as drought,cold and high salinity restricted greatly plant growth and productivity,they are the fatal factors induce productivity of alimentary crop.With the population keeping increasing and decreasing of cropland area,how to use the desert and aline lands is an very improtant problem.Undoubtedly,improvementing species through plant genetic engineering technology making crops to take possession of higher resistance to osmotic stress,that is a utility way which can solve this problem.At present,how to increase plants ability of resisting osmotic stress have been become a hot spot of plant genetic engineering on osmotic stress,nevertheless,screening risistance osmotic key genes from massive osmotic stress genes become a criticality aspect of plant genetic engineering on osmotic stress.In this study,we screening predicted genes by real-time PCR based on the predicted osmotic stress related genes from arabidopsis,which were obtained by bioinformatics method,we transgenic Osmotic Stress Responsible Genes which were obtained by real-time PCR in to tobacco,to validate gene function.2 new genes have resistance osmotic stress function were got.our study not only providing experimental evidence for previous genes function prediction,but also providing important genes for plant genetic engineering on osmotic stress.The main results were summarized as follows:1.Real-time PCR screening Osmotic Stress Responsible GenesThe expression of 8 arabidopsis genes were identificated by real-time PCR,we obtain 4 up-regulation express genes at osmotic stress,designating them AtMYB126,AtIMP,AtDBP1,AtDBP2 respectivly.2.Construction of plant expression vectorWe constructed four plant expression vectors:pBMYB,pBIMP,pBP1,pBP2 which regulated by CaMV35S promoter,and then the vectors are translated into Agrobacterium BLA4404 for tobacco transformation.3.Transformation mediated by Agrobacterium TumefaciensThe 4 gene above which were regulated by promoter CaMV35S,were transferred into tobacco "Longjiang 911".And each combination all obtained some resistant seedlings.4.Screening of transgenic plants and Identification of resistent seedlings Discs from resistent seedlings screening on MS medium contain Km,then we can obtain the seedings under the same genetic background.There were 21 differentiated lines in 24 lines with AtDBP1,20 differentiated lines in 24 lines with AtDBP2,25 differentiated lines in 30 lines with AtMYB126,25 differentiated lines in 30 lines with AtIMP.Seedings obtained by secondary screening were accepted detection of salt resistence and droughe:In 11 salt tolerance lines with AtDBP1,there were 8 lines have drought tolerance;In 11 salt tolerance lines with AtDBP2,there were 9 lines have drought tolerance.Unfortunatly transgenic lines with AtMYB126 and AtIMP did not demonstrate significant higher tolerance to stress.5.Molercular identification of resistance plantsResistance plants were detected by PCR and real-time PCR.In 12 salt resistance lines,there were 10 PCR positive plants.We selected 5 lines from them randomly,and 2 over-expression AtDBPI lines were obtained;In 11 salt resistance lines,there were 8 PCR positive plants.We selected 5 lines from them randomly,and 4 over-expression AtDBP1 lines were obtained.6.Identification of resistant plants physiological indexResistant plants physiological index was detected,resistant plants' conductance under salty stress was more stationary than control;chlorophyll in transgenic tobacco under salty stress was higher than control.