Dissertation > Agricultural Sciences > Gardening > Vegetable gardening > Cabbage

Molecular Cloning and Overexpression of Selenocysteine Methyltransferase from Broccoli (Brassica Oleracea Var. Italica)

Author LiBin
Tutor ZhengJinGui;HuangKe
School Fujian Agriculture and Forestry University
Course Biochemistry and Molecular Biology
Keywords Broccoli Sulforaphane Selenium methyltransferase (COMT) SMT Overexpression RNAi
CLC S635
Type Master's thesis
Year 2009
Downloads 75
Quotes 2
Download Dissertation

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 .

Related Dissertations
More Dissertations