Cloning And Expression Of The Abi5Gene Rfom The Arabidopsis Thaliana In E. ColiBL21

Plants are exposed to abiotic environmental stress such as drought, high salt andlow temperature, which cause seriously adverse effect on the growth and developmentof the plant. Plants have evolved to perceive different stress signals from theirsurroundings, to integrate them and to respond to these different stresses by causing aseries of physiological and biochemical reaction. The products of genes induced bydrought stress can be classified into two groups: those that directly protect againstenvironmental stresses, and those that regulate gene expression and signal transductionin the stress responses. The first group includes proteins such as late embryogenesisabundant proteins, antifreeze proteins and detoxification enzymes. The second group ofgene products includes transcription factors, such as bZIP，MYC，DREB, which playkey roles in modulating the transcription and expression of genes. Plants can chang thestress signals into the internal informationm of cells by initiating the complex singaltransduction pathway. The transcription factor can be activated through a series ofphosphorylation cascade to have the related gene transcription promoted. Abscisic acid(ABA) is a phytohormone and mediates the response and adaptation of higher plants tovarious environmental stresses during vegetative growth. ABI5is an ABA-responsedprotein of bZIP family.It can induce the transcription and expression of the reisistancegenes,which plays an important role on signaling, transcription control, regulationduring the embryonic development stage.The E. coli has been widely used in gene engineering for producing recombinantprotein with the advantages of short period, easy control and clear genetic background.There are several problems for prokaryotic expression which still remained unsatisfiedsolved, for example, the low solubility, relative low expression level and easydegradation of the products. In this research, Arabidopsis thaliana abi5gene was clonedand the prokaryotic expression plasmid pET32a-ABI5was constructed. Then thepET32a-ABI5plasmids were transformed into E. coli BL21for expression andOptimization of expression conditions. The main research results are as following:1. Cloning of abi5gene amplified from Arabidopsis thaliana: A pair of primerswas designed according to digestion sites in plasmid pET-32a and the abi5genesequence published by NCBI. The DNA fragment of abi5gene was amplified byRT-PCR which cloned into pMD-T Vector，then the recombinant was constructed and named pUC19-ABI5. The sequencing result showed that the whole length of the abi5gene of Arabidopsis thaliana was1323bp and coded for440amino acids.2. The construction of ABI5expression vector and transformation of reformedBL21Star（DE3）: The recombinant of pUC19-ABI5was digested with EcoR I. Afterpurification, the abi5-containing fragment was inserted into cloning site of pET-32adigested with EcoR I and pET32a-ABI5was constructed. According to the sequence ofcloned abi5, we chosed the reformed BL21Star（DE3）to be transformed and constructthe BL21/pET32a-ABI5prokaryotic expression strain.3. Optimization of Prokaryotic Expression：Screened positive clones for expressionwhose result of SDS-PAGE analysis showed the solution ABI5fusion protein wasexpressed efficiently, whose molecular weight is approximately67kD. We have theinduction conditions of BL21/pET32a-ABI5optimized, such as concentration of IPTG,temperature and induction time. The target fusion protein was mainly expressed in thesoluble form under all of the designed conditions. The soluble expression level appearedno obviouse difference both in the set concentrations of IPTG and the temperatures butdecreased with the time prolongation.The optimized condition was determined asinduction for3h with0.3mmol/L IPTG at30℃.