Plant promoter driven heterologous expression of HMW glutenin gene(s) subunit in E. coli.
Study of the structure of high molecular weight (Mr) glutenin subunits and their interaction has been limited by the difficulty of isolating single subunits in large amounts from the complex mixture of gluten wheat endosperm. One way to overcome this problem is the expression of high Mr glutenin subunits in heterologous systems. These systems have the additional advantages that, specific gene modifications can be made and the new gene constructs can quickly be expressed. HMW glutenin gene subunits, associated with better dough quality, were isolated from wheat (T. aestivum var. PBW343) using allele specific primers (AS-PCR) designed on allelic gene sequences. Three glutenin genes 1Ax1, Dx5 and Dy10 of sizes 1.8, 2.0 and 1.47 kb were cloned and sequenced. Probable secondary structure of HMW glutenin protein was predicted using online ExPASy server. HMW glu Dx5 and Dy10 gave significant similarity (more than 90%) to the available database but, 1Ax1 didn't show significant similarity. Glutenin genes were further subcloned in expression vector pCAMBIA-1304 driven by CaMV35S promoter and their expression was observed in Escherichia coli. The transcription was confirmed by Reverse transcriptase PCR (Rt-PCR) and sequencing. The sequences of HMW glu genes showed 100% homology with cloned HMW glutenin genes. Although initial western blotting using polyclonal antibodies against glutenin protein has strongly indicated its expression at the translation level, it is needed to be further confirmed through purification and sequencing of proteins.
Pandey
Ruchi
R
Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture & Technology, Pantnagar, 263 145, India.
Mishra
Avinash
A
Garg
G K
GK
D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D005822:Genetic Vectors; D005983:Glutens / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014908:Triticum / Q000235:genetics*
D021122:Protein Subunits; D005983:Glutens; C037302:glutenin