Cloning and Prokaryotic Expression Activity Determination of A BI5 Gene from Picea schrenkiana
ABI5,also known as abscisic acid-insensitive protein 5,is an important transcription factor in ABA signaling pathway,which plays a coordinating role in the regulation of seed development and germination,plant growth,and stress.At present,most of the studies are on Arabidopsis thaliana,but there are few reports on woody plants such as Picea schrenkiana.In this study,the cDNA of Picea schrenkiana was used as the template for PCR amplification,and the ABI5 sequence of Picea schrenkiana was obtained.The total length of ABI5 was 1 071 bp,encoding 352 amino acids.The recombinant plasmid pET-24a-ABI5 was obtained by cloning ABI5 gene into pET-24a expression vector.IPTG concentration,induction time,medium type and incubation temperature were optimized,and the optimal culture condition was LB medium,the final concentration of IPTG was 0.2 mmol/L,and the incubation temperature was 20 ℃ for 8 h.The purified products were detected by SDS-PAGE and most of the proteins were found to exist in the form of inclusion bodies.Some of the proteins were renaturated and their activity was verified by malachite green chromogenic reaction.
Picea schrenkianaABI5Molecular cloningProkaryotic expressionRenaturation of inclusion bodies
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