Cloning a Full—length cDNA Encoding UDP—glucose Pyrophosphorylase from Amorpha fruticosa by PCR—based Methods

A method based on degenerate Oligo-primed polymerase chain reaction(PCR) and randomamplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described.An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase(UGP),a key enzyme producinng UDP-glucose in the synthesis of sucrose and cellulose,is cloned by using this method.We design 5‘ RACE rpimers based on UGPA1 fragment ,which obtains from degenerate PCR.Inverse PCR and nested PCR enable cloning of the remainder 5‘ and 3‘ end fragments of the gene.The deduced amino acid sequence xhibits significant homology with the other UGP genes cloned.This method is more simple and inexpensive than screening cDNA library,and can be easily adapted to clone other genes.

Cloning a Full-length cDNA Encoding UDP-glucose Pyrophosphorylase from Amorpha fruticosa by PCR-based Methods

A method based on degenerate Oligo-primed polymerase chain reaction (PCR) and random amplification of cDNA end (RACE) PCR for cloning a full-length cDNA is described. An Amorpha fruticosa cDNA clone encoding UDP-glucose pyrophosphorylase (UGP), a key enzyme producing UDP-glucose in the synthesis of sucrose and cell ulose, is cloned by using this method. We design 5' RACE primers based on UGP A1 fragment, which obtains from degenerate PCR. Inverse PCR and nested PCR enable cloning of the remainder 5' and 3' end fragments of the gene. The deduced amino acid sequence exhibits significant homology with the other UGP genes cloned. This method is more simple and inexpensive than screening cDNA library, and can be easily adapted to clone other genes.