Molecular Cloning And Overexpression Of Selenocysteine Methyltransferase From Broccoli (Brassica Oleracea Var. Italica)

Selenocysteine methyltransferase (SMT), specifically methylates selenocysteine (SeCys) to produce the nonprotein amino acid Se-methyl selenocysteine (SeMSC) and played key role of removing toxic effect at higher selenium levels. Because selenium and sulfur have similar chemical properties and same metabolic pathway, which exist competitive inhibition and interactions between their corresponding metabolites. Broccoli not only has rich sulfur-containing compounds characteristic, but also has the selenium-enriched characteristic, its important activity compound-Sulforaphane, which is synthesised by sulfur-containing amino acid (methionine), due to the sulfur is replaced by selenium in metabolic process, selenium methionine in selenium metabolic pathway instead of methionine in sulfur metabolic pathway, thus affecting metabolic synthesis of the Sulforaphane.In order to study the effect of SMT gene on the Sulforaphane, this gene was cloned and overexpressed. The results are as follow:1. SMT gene cDNA sequences from broccoli (Brassica oleracea var. italica) young leaf were amplified through RT-PCR and the coding sequences of this gene were analyzed. The results demonstrated that the coding sequences of cloned SMT is 1041 bp which shared 99% homogeneity to the sequences of AY817737 genes.2. Overexpression and RNAi vectors of SMT gene were constructed. Overexpression vector was constructed, in which the coding sequces of the gene was placed under the 35S promoter of pBI121. In addition, a 473 bp fragment of Selenocysteine methyltransferase gene was cloned from Brassica oleracea var. italica by RT-PCR and inserted into the two sides of the intron in pJM007 vector in a reverse way to produce the SP(RNAi) cassette. This cassette was excised from pJM007 by Pst I endonuclease and inserted into the binary vector pCAMBIA1301 to produce SP(RNAi) vector.3. pBI121-SMT was transferred into Agrobacterium tumefaciens LBA4404 and then transformate to the broccoli, 20 transgenic plantlets were obtained under the selection of kanamycin. 8 transgenic lines were identified by PCR, and Selenocysteine methyltransferase genes were effectively overexpressed in the transgenic plants by the real time fluorescence quantitative PCR analysis.