Cloning And Expression Analysis Of Oligopeptide Transporter Gene Of Maize (Zea Mays L.)

Improving nitrogen use efficiency of crops plays a pivotal role in ensuring global food security.The researches focusing on crop nitrogen utilization have been conducted extensively.The results of plant molecular physiology demonstrated that Oligopeptide transporter (OPT) was involved in the transporting and utilization of plant nitrogen and had a significant impact on plant nitrogen utilization. In this study, with elite maize inbred line Ye478 and its three introgression lines IL8, IL26 and IL65 as materials, Maize Oligopeptide transporter genes were cloned by RT-PCR and analyzed by Bioinformatic.Expression analysis was carried out by subcellular localization and semi-quantitative PCR. The main research contents and results are as follows:1. The cDNA of Oligopeptide transporter gene from nitrogen-sensitive maize inbred lines named ZmOPT0212 (Accession number: HM021150) was isolated by RT-PCR,with primers designed according to the sequence of Oligopeptide transporter full-length gene in maize inbred line B73 and rice. The open reading frame of ZmOPT0212 gene was 2151 bp in length and encoded a polypeptide of 716 amino acid residues. The estimated molecular weight of the gene was 77.4kD and the isoelectric point of the putative protein was 8.87respectively.2. The predicted secondary structure composition for the protein had about 43.85% alpha helixes, 13.27% extended strand, 3.91% beta turn and 38.97% random coil. Hydrophobicity/hydrophilic analysis indicated that the ZmOPT0212 had 14 hydrophobic segments containing 12 potential transmembrane segments, and the ZmOPT0212 had no signal peptide and was located on plasma membrane. Conserved domain prediction showed that encoded protein belonged to OPT protein family and had the function of transcription,transcription regulation,signal transduction, metal ion transport,transport and binding by gene ontology prediction.Blast results and the phylogenetic tree analysis showed that peptide encoded by ZmOPT0212 of maize inbred lines Ye478 had high identity with the Oligopeptide transporter of the rice, Arabidopsis thaliana, Populus trichocarpa, and Ricinus communis , with which the similarity 99%,96%,92% and 92% ,respectively.3. Through Subcellular localization,the fusion expression vector 35s-GFP-ZmOPT0212 was successfully constructed. In order to observe its position, the plasmid was inserted into the onion epidermal cell by the gene gun. The result showed that the ZmOPT0212 located in the cytomembrane.4. Semi-quantitative analysis showed that, ZmOPT0212 gene expressed both in young leaves and roots in non-stress condition. Also, the ZmOPT0212 gene expressed in Ye478 and its inbred lines in both leaves and roots under abiotic stress condition through semi-quantitative RT-PCR analysis. Compared with Ye478, the expression ZmOPT0212 gene had an upward trend in IL26, but the other two introgression lines increased rapidly in the early stages, then stabilized or declined under nitrogen stress.