关键词: 甘蓝型油菜, 油体钙蛋白, BnClo1基因, 原核表达, pTYB12

Abstract:

【Objective】 BnClo1gene cloned from Brassica napus was expressed in Escherichia coli ER2566. 【Method】 A cDNA fragment about 750 bp was amplified from the total RNA of rape seeds by reverse transcription PCR (RT-PCR) with a pair of specific primers based on the sequences of the BnClo1 gene. The recombinant prokaryotic expression vector pTYB12-BnClo1 was constructed by inserting the cDNA fragment encoding the mature peptide of caleosin into the prokaryotic expression vector pTYB12, and then transformed into E. coli ER2566 (DE3). 【Result】 Sequence analysis showed that the fragment length was 768 bp containing a full coding region of 738 bp encoding 245 amino acid residues with a molecular mass of 28.1 kD. The SDS-PAGE electrophoresis analysis showed that the best expression was induced by 20℃ and 4 mmol?L-1 IPTG, under which a relative molecular weight of 83.1 kD recombinant protein intein-caleosin was produced. 【Conclusion】 A BnClo1gene of rape was cloned and optimizing expressed in E. coli. These results should provide a foundation for further purifying and identifying target protein and function study of rape caleosin protein.

Key words: rape (Brassica napus), caleosin, BnClo1 gene, prokaryotic expression, pTYB12