Cloning Of Transcription Factor Regulating Stress-Responsive Related Gene Of Wheat

A GAL4AD cDNA fusion expression library and a phage cDNA library were constructed using mRNA isolated from wheat seedling leaf dehydrated for 2 hours.The liter of the GAL4 cDNA expression library is l?06pfu/ml,and 103 positive clones selected randomly were digested with EcoR I and Xho I to evaluate the quality of cDNA library.The size of the inserted segment ranges from 200 to 6000bp with an average of 2000bp.The yeast one-hybrid system was applied to screen the GAL4 cDNA library.One positive cDNA clone was named Y82.Searching the database using the ammo acid sequence of Y82 revealed that the homology between Y82 and plants protein kinase is high.The deduced protein sequence contains a complete open reading frame(ORF) of 762bp encoding 254 amino acids.Blast analysis was performed against databases in NCBI,and the result showed that the homology is 76% between Y82 and protein kinase(Zea mays) with leucine-rich repeat transmembrane protein kinase 2(ltk2),67%Glycine max receptor-like protein kinase l(RLKl),and 65 % Arabidopsis thaliana wall-associated receptor kinase genes(wakl).The Y82 was considered as a protein kinase-like gene.Expression of RLK1 was induced by Low temperature,drought, and high salinity,and RLK1 can perceive environmental signals and transmit them to cellular machinery to activate adaptive responses.So we deduced that research of Y82 will certainly expedite our understanding of stress signaling mechanisms in wheat.The titer of the phage cDNA library is lxl06pfu/ml.A specific band amplified from wheat was used as probe to screen cDNA library.The first screening,we obtained 44 positive clones,then used these clones to perform the second screening, and a postive clone,W6 was successfully obtained.Blast analysis was performed against databases in NCBI,and the result showed that the homology is 52 % between W6 and EREBPl(Oryza sativa) transcription factor,43 % Nicotiana tabacum C-repeat/DRE-binding factor.