Cloning Key Enzyme Genes (NAD-ME,PEPC) Of C₄ Photosynthesis From Amaranthus Hypochondriacus L. And Function Prediction Of NAD-ME

Compared with C3 plant, C4 plant has evident growth advantage, higher rate of nutrition using and higher bio-yield in extreme condition (such as high light intensities, high temperatures and drought) because of efficient photosynthesis and non-photorespiration. Cloning the key enzymes of C4 photosynthetic pathway in Amaranthus hypochondriacus L. as one of C4 dicotyledon provides candidate gene for C4 genetic engineering.NAD/NADP-malic enzyme and phosphoenolpyruvate carboxylase (PEPC)are two key enzymes of C4 pathway. NAD/NADP-ME catalyzes the oxidative decarboxylation of 1-malate to yield pyruvate, CO2, and NADPH in the presence of a divalent cation. In C4 plants, NAD/NADP-ME release CO2 for fixation by Calvin cycle. In this study, a full-length cDNA encoding Amaranthus hypochondriacus L. NAD-ME was cloned by RT-PCR. The cDNA includes a 1872-bp open reading frame that encodes 623 amino acids. Blastn and DNAMAN analysis reveals that the nucleotide sequence has 75.1%-80.6% identity. Furthermore, the physico-chemical property of NAD-ME of Amaranthus hypochondriacus L.,including the conservative domain, hydrophobicity/hydrophilicity, potential transmembrane segment, signal peptide, intrinsically disordered structure and secondary structure of the hypothetical protein were analyzed. Codon bias of Amaranthus hypochondriacus L. NAD-ME was further analyzed by Codon W, CHIPS and CUSP programs for identifying codon bias. Moreover, the codon bias of Amaranthus hypochondriacus L.NAD-ME were compared with that of NAD/NADP-ME gene of other seven plants and two model organisms, Escherichia coli and yeast. The results showed that codon bias of NAD/NADP-ME of monocotyledons and dicotyledonous plants obviously remarked at some codons which were the A or T in the 3rd position of codons. Codon bias of NAD-ME of Amaranthus hypochondriacus L.was significantly different from those of E.coli and yeast genome.The result has important guidance for choosing appropriate recipient plants and protein exprssion system of NAD-ME gene of Amaranthus hypochondriacus L. PEPC catalyzes irreversible carboxylation of PEP in the presence of Mn2+ and Mg2+ to yield OAA. In this study, we cloned the genomic gene of PEPC of Amaranthus hypochondriacus L. firstly by PCR. According to Blast analysis, we found most of the cloned DNA sequences similar to that of PEPC gene of other plants. And the identity to the F.pringlei PEPC was 93%.Thus, we determinated the cloned fragment was Amaranthus hypochondriacus L. PEPC preliminarily.