Abstract: Full-length cDNA sequences of the LpP5CS gene, previously obtained using PCR amplification were used as a basis for developing a site-directed mutagenesis system of the Lolium perenne proline synthetase gene LpP5CS. The gene was used in Agrobacterium-mediated transformation technology to verify a functional mutant gene. A pair of primers was designed to the mutation sequence and was used with Pfu high-fidelity DNA polymerase and super-competent cells DMT in PCR amplification. The LpP5CSF128A mutant gene which contained the desired mutational sites was directly cloned into the eukaryotic expression vector pCAMBIA1300, for transformation into Arabidopsis for validation of gene function. The expected locus mutations had occurred in LpP5CS and changed the encoding codon No.128 from a phenylalanine residue (Phe or F) into an alanine residue (Alanine, Ala). This was proof that PCR technology had succeeded in site-directed mutagenesis of the LpP5CS gene. Four transgenic lines were obtained from the T₁ generation of transgenic Arabidopsis plants by PCR and RT-PCR. The T₂ generation of Arabidopsis plants were treated with 100 mmol/L NaCl solution for 7 d and the proline content of T₁ and T₂ transgenic plants were 4 262 and 5 623 μg/g FW respectively, higher than the 2 581 μg/g FW of wild-type strains. This indicated that transgenic strains can accumulate more proline.