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Cloning and Expression Analysis of Cyt b5 Gene Involved in Electron Transfer in Gossypium hirsuturm |
LIU Jin-zhi1,2, SI Huai-jun1, ZHANG Ning1, WU Jia-he2 |
1. Life Science Institute of Technology, Gansu Agricultural University, Lanzhou 730070, China; 2. State Key Laboratory of Plant Genomic, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China |
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Abstract On the basis of salt-stress related EST library from Gossypium hirsuturm L. A partial sequence fragment of cytochrome b5 proteins (Cyt b5) encoding gene was obtained through homologous comparison with other plants. The Cyt b5 gene was isolated by the 3',5'-RACE technology from G. hirsuturm, named GhCyt b5. The full-length cDNA of GhCyt b5 is 810 bp, containing a 402 bp ORF which encodes 134 amino-acid peptide. The relative molecular weight of GhCyt b5 protein is 17 kDa. The coding sequence fragment was amplified by PCR and cloned into vector pET32a to generate the pET32a-GhCyt b5 expression vector, which was transformed into BL21(DE3) for expression of recombinant protein. On the base of optimization of different inducible conditions, the results showed that soluble GhCyt b5 protein under the 28℃ and 1 mmol/L IPTG conditions could be obtained. The expression product of GhCyt b5 was purified by Ni2+ affinity column and identified by SDS-PAGE. Using extracted cotton cell material as reaction medium for in vitro electron transfer analysis, it can be found the GhCyt b5 involved in the electron transfer system and accepted electron to become oxidation state from reducing state. Ultimately, the Cyt b5 gene for the first time from G. hirsuturm was cloned and its participating in electron transfer system was confirmed, which provide a base for further dissecting function in biotic and abiotic resistance.
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Received: 15 January 2013
Published: 25 April 2013
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