甘蓝型油菜BnADC基因的克隆及原核表达

Cloning and prokaryotic expression of BnADC gene from Brassica napus L.

利用同源克隆和RACE(Rapid amplification of cDNA ends)技术从甘蓝型油菜中克隆到精氨酸脱羧酶(Arginine decarboxylase,ADC)基因cDNA全长序列,命名为BnADC。BnADC全长为2 649bp,包含344bp的5'非翻译区(5'Untranslated region,5'-UTR)、226bp的3'非翻译区(3'Untranslated region,3'-UTR)和2 079bp的完整开放阅读框(open reading frame,ORF),编码75.1kD的蛋白质。5'-UTR中含有12bp的uORF(Upstream open readingframe),编码MIRE 4个氨基酸。氨基酸同源性比对表明,BnADC蛋白与其他植物ADC蛋白具有很高的相似性,与芥菜的同源性高达93%;系统进化树分析表明,BnADC与芥菜、拟南芥的ADC亲缘关系较近。在获得全长cDNA的基础上,构建融合表达载体pET30a(+)-BnADC,转化大肠杆菌(Escherichia coli)表达菌株BL21(DE3),SDS-PAGE检测到一个约81.1kD的融合蛋白被E.coil表达,且融合蛋白主要存在于菌体沉淀中。

An arginine decarboxylase gene ADC,named BnADC,was isolated by homologous cloning and RACE technology using fresh bud of Brassica napus L.cv Zhongshuang 6.The full cDNA was 2 649bp including 344bp of 5′-UTR,226bp of 3′-UTR and 2 079bp of an open reading frame（ORF）.Also a small uORF encoding four amino acids,as MIRE,was found in 5′-UTR.Amino acid homology analysis indicated that BnADC protein was highly homologous to other ADC proteins which had 93% identity to that of Brassica juncea.Phylogenetic tree analysis revealed that BnADC was more related to those of Arabidopsis thaliana and B.juncea.Based on the full cDNA of BnADC,a fusion expression vector pET30a（＋）-BnADC was constructed,then transformed into Escherichia coli strain BL21（DE3）.SDS-PAGE electrophoresis analysis showed an 81.1kD fusion protein produced,which was mainly in the deposition of transformed E.coli debris.