Molecular Cloning And Prokaryotic Expression Of Myrosinase Gene From Brassica Alboglabra L. H. Bailey

Sulforaphen, one kind of isothiocyanates, has been identificated the bestanticarcinogenic activity in vegetable. Glucoraphanin is the precursor of sulforaphen,its content in Brassica alboglabra L. H. Bailey is the highest in Brassica vegetable.Isothiocyanates act as repressor and inhibitory factor in the mechanism of cancerchemoprevention.They also have functions of antibiotic, antisepsis and plant diseasesand insects pests resisting. Myrosinase is the key enzyme of isothiocyanates synthesispathway in plants. The synthesis of natural isothiocyanates is by means of catalyzingglucosinolates by myrosinase.In this paper, the full-length cDNA of myrosinase gene was coloned from thelaminae of Brassica alboglabra L. H. Bailey by RT-PCR and RACE. The openreading frame (ORF) of myrosinase gene was subcloned into prokaryotic expressionalvector pET28-a(+) to construct recombinant plasmid. The recombinant plasmid wastransformed into E.coli strain B121 and induced by IPTG to express. The majorexperiment results of this research are as follow:1．Molecular cloning and characterization of myrosinase gene.The total RNA wasextracted from the laminae of Brassica alboglabra L. H. Bailey. A pair of specificprimers were designed according to the conserved nucleotide sequence of differentplants. 771bp sequence was obtained by RT-PCR. By application of RACE, the fulllength cDNA was 1807bp, including an open reading frame(1647bp) which encoded548 amino acids. The gene was named as Myrshjg. Bioinformation analysis suggestedthat full length cDNA sequence of Myrshjg showed 97%, 97%, 96%, 92%, 93%, 91%,87% similarity to Brassica napus, Brassica rapa , Brassica rapa.subsp.pekinensis,Brassica Juncea, Raphaus Sativus, Sinapis alba and Eutrema Wasabi respectively,myrosinase amino acid sequence of Brassica alboglabra L. H. Bailey has 99%, 98%,98%, 93%, 94%, 90% and 80% similarity with Brassica napus, Brassica rapavar.parachinesis, Brassica rapa.subsp.pekinensis, Brassica Juncea, Raphaus Sativus,Sinapis alba and Eutrema Wasabi respectively. Based on the aboved results, it couldbe inferred that Myrshjg should be myrosinase gene. 2．Prokaryotic expression of myrosinase.By inserting the open reading frame (ORF)of myrosinase gene into prokaryotic expressional vector pET28-a to constructrecombmant plasmid. The recombinant plasmid was transformed into E.coli strainB121 and induced by IPTG to express.The result of SDS-PAGE electrophoresisindicated that myrosinase gene had expressed in E.coli and the molecular mass ofrecombinant protein was about 65 kD.Therefore the result proved that Myrshjg wasmyrosinase gene and it could express in E.coli.