Cloning、Overexpression Vector Construction Of1-2Gene ORF And Its Genetic Transformation

Drought causes an effect in reduction of wheat yield among numerous abiotic stresseswhich makes the research of plant drought stress and relative stress-resistant mechanism hotspots in international study. Under drought stress, the plant could express a series of droughttolerance protein to response to stress and adapt drought stress through drought-resistancemechanism.Drought stress induces many expression of genes including Dehydrin genes. Dehydrin ispart of Late embryogenesis abundant protein-Ⅱ subfamily. The classification of dehydrins isbased on their different conserved domains(Y, S and K segments). Dehydrins are heat-stableprotein, it could be induced by drought, ABA, cold, hot, and salinity stress. Dehydrin could beinduced by adversity stresses among many proteins response to the stress. Dehydrin showshighly hydrophilic that it could prevent water deficit in plant cells, protect hydration systemof cytomembrane and inhibit shrinking of bilayer lipid membranes through binding withmembrane lipid of cells to reduce the damage in biomembrane. In this study, we cloned thewzy1-2gene in Wheat Zhengyin1#, use leaf dish transformation induced by Agrobacteriumtumefaciens to research genetic transformation in tobacco and successfully obtainedtransformation tobacco of wzy1-2gene that laid the foundation of wzy1-2gene functions.The results of the experiment are as follows:1. Using the primers designed by wzy1-2virtual sequence, we cloned wzy1-2ORF Thewzy1-2ORF was cloned in789bp, which coding262aa. The bioinformatics analyses indicatedprotein wzy1-2maybe the nucleoprotein, possessing8hydrophobic regions and17phosphorylation sites.2. Successfully constructed the plant overexpression vector pBI121-wzy1-2, and thevector was transformed into tobacco via Agrobacterium tumefaciens induced leaf dishtransformation method, gaining Kan resistance transformed tobaccos. Use PCR、semi-quantitative PCR and sequencing detecting T0plant to ensure the wzy1-2gene istransformed into genome of tobacco and transcribing normally.