Molecular cloning and characterization of P5CS gene from Jatropha curcas L.
Δ 1-Pyrroline-5-carboxylate synthetase, (P5CS) is a bifunctional enzyme (EC 22.214.171.124/126.96.36.199) that catalyses the first two steps of glutamate pathway in proline biosynthesis in plant. The JcP5CS was cloned from the leaves of Jatropha curcas L. The lengthen of the cDNA of JcP5CS was 2675 bp, containing a 2148 bp open reading frame, a 117 bp 5’-untranslated region and a 410 bp 3’-untranslated region. The open reading fragment (ORF) encoded a 715 amino acid polypeptide with the molecular weight of 77.54 kDa and the pI value of 6.11. JcP5CS was composed of N-terminal Glutamate-5-kinase (G5K, ProB) and C-terminal glutamic-γ-semialdehyde dehydrogenase (GSA-DH, ProA) domains. The conserved Glu-5-kinase, GSA-DH domains, conserved leucine zipper and the putative ATP and NAD(P)H-binding sites was also found. The JcP5CS protein was successfully expressed in Escherichia coli and showed high enzymatic activities. The real-time quantitative PCR results showed that the JcP5CS was induced by drought and salt stress, but not cold stress.
Key words: Δ 1-Pyrroline-5-carboxylate synthetase, proline, Jatropha curcas L., expression, molecular cloning, real-time quantitative polymerase chain reaction (RT-PCR).