Molecular Cloning and Overexpression of Selenocysteine Methyltransferase from Broccoli (Brassica Oleracea Var. Italica)
|School||Fujian Agriculture and Forestry University|
|Course||Biochemistry and Molecular Biology|
|Keywords||Broccoli Sulforaphane Selenium methyltransferase (COMT) SMT Overexpression RNAi|
Selenium methyl transferase (SMT) can seleno cysteine ??-specific methylation of non-protein amino acids - selenium methyl selenomethionine cysteine ??, and play a key role in plant resistance to selenium toxicity effect efficient . Competitive inhibition exists between their respective metabolites with similar chemical characteristics and the same metabolic pathways as selenium and sulfur , selenium and sulfur the interaction relationship broccoli both rich in sulfur compounds characteristic , and also have selenium-enriched characteristics of its active ingredient - sulforaphane (Sulforaphane) is synthesized from methionine selenium selenium in the metabolic process to replace the position of the sulfur , selenium metabolic pathways of sulfur-containing amino acids ( methionine ) the substituted sulfur metabolic pathways methionine , thus affecting Sulforaphane metabolic synthesis . In order to study the SMT gene regulatory role of sulforaphane , we SMT gene cloning and overexpression , and obtained the following results : 1 . Selenium methyl transferase cloned from broccoli leaves by RT-PCR method ( Selenocysteine ??methyltransferase, SMT) gene coding sequence, a length of 1041 bp. The homology comparison results show that : the SMT gene have been reported in GenBank (GenBank No.AY817737) 99 % homology . Building selenium methyltransferase gene overexpression and RNAi vectors . PBI121 plasmid was constructed by the CaMV35S promoter regulation SMT gene expression vector pBI121- SMT ; also cloned SMT gene a 473 bp section of the highly conserved sequence SP , and the forward and reverse to the insertion into the vector pJM007 intron sides of the elements forming a SP (RNAi) by Pst I digestion and cloned into the plant expression vector into pCAMBIA1301 vector pCAMBIA1301 SP (RNAi) is formed . By the freeze-thaw method the pBI121-SMT carrier transferred into Agrobacterium tumefaciens ( Agrobacterium tumefaciens ) strain LBA4404 harboring . Tumefaciens LBA4404, the genetic transformation broccoli , 20 regenerated plants obtained after kanamycin with pBI121-SMT carrier . After PCR, access the 8 transgenic renewable strains by quantitative PCR analysis showed that selenium methyltransferase gene expression in transgenic plants increased significantly .