Cloning Of MhP5CS, MhOAT In Malus Hupihiesis (Pamp)Rehd. And Their Response To Cadmium Stress

Annual seedlings of Malus hupehensis (Pamp)Rehd were used as materials in this experiment. Two genes which were key enzymes in the proline synthesis in plants have been cloned, such asâ–³¹-pyrroline-5-carboxylate synthetase (P5CS), ornithineδ-aminotransferase (δ-OAT). We discussed the principal way of the proline biosynthesis under heavy metal stress. The effects of exogenous polyamines and NO in alleviating the damage under heavy metal stress were studied. The results were as follows: 1. Primer pairs were designed based on highly conserved nucleotide sequences of otherplant genes previously submitted to the Genbank database. We have clonedâ–³¹-pyrroline-5-carboxylate synthetase and ornithineδ-aminotransferase genes using RT-PCR and RACE technology. We named these two genes MhP5CS, MhOAT, and submitted them in Genbank, the accession numbers were JF742986 and JF742987 respectively. The full-length cDNA of MhP5CS was 2470bp, and the ORF of MhP5CS encoded a 718-amino acid polypeptide. The relative molecular weight was 77.78kD and isoelectric point was 6.26. The full-length cDNA of MhOAT was 1807bp, and the ORF of MhOAT encoded a 478-amino acid polypeptide. The relative molecular weight was 52.09kD and isoelectric point was 8.06.2. Bioinformatics analysis indicated that MhP5CS protein was a hydrophilic protein, which contained five speculated transmembrane(TM) segments. The 150¹68aa TM segment has a strong transmembrane domain from outside to inside. Sequence comparison showed that the MhP5CS protein was closer to that of Euonymus japonicus and Jatropha curcas. MhOAT protein was also hydrophilic protein, which contained seven speculated transmembrane(TM) segments. The 286³05aa TM segment has a strong transmembrane domain from outside to inside. Sequence comparison showed that the MhOAT protein was closer to that of Euonymus japonicus and Jatropha curcas.3. It was measured that the content of proline (Pro), the expression of MhP5CS and MhOAT, the activities ofâ–³¹-pyrroline-5-carboxylate synthetase (P5CS) and ornithineδ-aminotransferase (δ-OAT) in Malus hupehensis Rehd. var. pinyiensis Jiang (tea crabapple) seedlings under cadmium sulfate (CdSO4) stress with different concentration and different time, these results showed that the expression of MhP5CS and the content of Pro increased along with the increasing of Cd concentration, but OAT has no obvious changes.4. It was measured the effect of exgnous NO and it's inhibitor on biosynthesis of Pro, these results indicated that NO can promote the biosynthesis of proline in Malus hupehensis under cadmium stress.5. It was measured the effect of exgnous spermine, spermidine and it's inhibitor on biosynthesis of Pro, these results indicated that spermine can promote the biosynthesis of proline in Malus hupehensis under CdSO₄ stress.