Construction Of OsbZIP81 Transgenic Rice Plants And Production Of Restriction Enzyme I-SceI

This study contains two parts: 1.Construction of OsbZIP81 transgenic rice plants and preliminary study on the interaction between OsbZIP81 and OsIMPa4;2.Production of restriction endonuclease I-SceI.Through Agrobacterium-mediated genetic transformation,T-DNA containing the foreign gene can be integrated into rice genome,and stably inherited.Previous studies have shown that the AtIMPa4 plays an important role in T-DNA Complex transpotation into plant,AtVIP1 plays an important role in T-DNA integration into the Arabidopsis genome.AtVIP1 is a bZIP transcription factor and can response to abiotic stress by regulating the downstream genes.We studied the rice homologous gene OsbZIP81 of AtVIP1.The results are as follows:1.OsbZIP81 belongs to rice bZIP IX subfamily.It contains three transcripts.By sequence comparison,we found that AtVIP1 and rice bZIP IX subfamily are highly conserved in the bZIP motif,OsbZIP81 has a high sequence similarity with AtVIP1.2.We constructed OsbZIP81 overexpression and CRISPR knockout transgenic rice plants.The expression level was significantly higher in the OsbZIP81 overexpression plants and lower in the CRISPR knockout plants than that of wild types.3.OsIMPa4 is the orthologous gene of AtIMPa4 in rice.Yeast two-hybrid and BiFC results showed that OsIMPa4 interacted with the three OsbZIP81 transcripts-encoded proteins.Restriction endonuclease is a very important tool enzyme in moleular biology.I-SceI is a restriction enzyme encoded by the mitochondrial intron of Saccharomyces cerevisiae.Its recognition site is 18 bases and non-palindromic.The cohesive ends produced by I-Sce I are asymmetric.Futhermore,the plant and animal genomes contains none or very few I-Sce I sites.Therefore,I-SceI is very suitable for detection of large DNA inserts of BAC libraries and large insert exchange between different vectors.In this study,the recombinant endonuclease I-SceI was obtained by gene synthesis,cloning,expression and purification.The purified GST-I-SceI protein has a concentration of 590 mg/L and an enzymatic activity of 0.2 U/?L.The specific activity of GST-I-SceI was 340 U/mg.