芥蓝黑芥子酶基因的克隆与原核表达  

Cloning and prokaryotic expression of myrosinase gene from Chinese kale(Brassica oleracea L. var. alboglabra Bailey)

作　　者：谢丽雪[1] 黄科[1] 李宾[1] 黄志伟[1] 郑金贵 

机构地区：[1]福建农林大学农产品品质研究所

出　　处：《福建农林大学学报（自然科学版）》2008年第5期501-505,共5页Journal of Fujian Agriculture and Forestry University:Natural Science Edition

基　　金：国家重大基础研究前期研究专项(2005CCA01300);福建省科技厅重大专项(2005NZ1022);国家自然科学基金(30600415);福建省科技创新平台建设项目(2007S1001);国家科技项目备案(F2005CCAO1300)资助

摘　　要：根据GenBank上已公布的植物黑芥子酶基因序列设计引物,采用RT-PCR和3′/5′-RACE相结合的方法,从芥蓝中克隆出黑芥子酶基因全长cDNA序列.序列全长为1832 bp,开放阅读框为1647 bp,编码549个氨基酸,含有糖基水解酶家族Ⅰ(Glyco_hydro_1)活性位点,GenBank登录号为DQ767973.该核苷酸序列与十字花科其他植物的黑芥子酶基因序列具有较高的同源性,同油菜、萝卜、芥菜和大白菜的同源性达90%以上.利用pET28-α(+)构建芥蓝黑芥子酶基因的原核表达载体,转入大肠杆菌BL21,经IPTG诱导表达,SDS-PAGE检测表明表达蛋白的分子质量约为65 ku.A pair of high conserved primers were designed according to the cDNA sequences of myrosinase genes published in Gen- Bank. The full-length cDNA of myrosinase gene was coloned from Chinese kale using RT-PCR and 3 ＇/5＇ RACE. Nucleotide sequence analysis results showed that the full sequence of myrosinase gene was 1832 bp, including 1647 bp of ORF. The ORF encoded a peptide of 549 amino acid residues and had Glyco_hydro_1 site. This sequence has been registered on GenBank and the Gen- Bank accession number !s DQ767973. Compared with the sequences of myrosinase gene from other reported Cruciferous species, the result of blast analysis showed that they shared with high homology. It was more than 90% similarity to Brassica napus, Raphanus sativus, B. juncea and B. rapa subsp. To characterize its function, the myrosinase expression vector was constructed with pET-28α （ ＋ ） and transformed into Escherichia coil BL21. The SDS-PAGE result showed that a 65 ku recombinant protein was expressed and separated after indeced by IPTG.

关 键 词：异硫氰酸盐 黑芥子酶 芥蓝 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学]