芥蓝黑芥子酶基因的克隆与原核表达

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.

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

A pair of high conserved primers were designed according to the cDNA sequences of myrosinase genes published in GenBank.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 GenBank accession number is 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 coli BL21.The SDS-PAGE result showed that a 65 ku recombinant protein was expressed and separated after indeced by IPTG.