Isolation And Expression Analysis Of An Alternative Oxidase Gene, NgAOX1α, From Nicotiana Glutinosa

Plant mitochondria possess a bifurcated electron-transport chain. In addition to the cyanide-sensitive cytochrome (cyt) respiratory pathway found in all eukaryotes, plants have a second, cyanide-insensitive alternative pathway. Electron flow via the cyt pathway is coupled to ATP production, whereas electron transfer through the alternative pathway branches from the cyt pathway at the ubiquinone pool and reduces oxygen to water without conservation of energy in the form of ATP, which is lost as heat. This reaction consists of a single step that is solely mediated by alternative oxidase (AOX). AOX is the terminal oxidase of the cyanide-insensitive alternative pathway in the mitochondrial inner membranes. AOX proteins are found in everyexamined species and in almost every plant organ. The cyanide-resistant respiratory pathway and the biological function of AOX are always the hot study in plant physiological research. Except for producing heat energy during anthesis in some plant (benefit for pollination), it is still an open question for other biological functions of AOX and the tertiary structure of AOX. In this thesis, we cloned an AOX gene from Nicotiana glutinosa (NgAOX1a) and a series of studies have been conducted on the isolation, sequence and expression analysis of NgAOX1a. At the same time, we also isolated the promoter of NgAOX1a from Nicotiana glutinosa, and contructed the expression vectors for delection analysis. It provides a basis for further investigation of the cis-acting elements. The main results are as follows:1. A novel gene, named NgAOX1a (GenBank Accession No. EF523518), was isolated from Nicotiana glutinosa by reverse transcription-PCR (RT-PCR). The full-length cDNA of NgAOX1a was 1448 bp, including a 1062 bp open reading frame (ORF), a 124 bp 5′untranslated region (UTR) and a 262 bp 3′UTR. The ORF encodes a 353-amino acid protein which contains two conserved cysteine residues, four iron binding motifs, fiveα-helix regions and six conserved histidine residues. Alignment analysis showed that NgAOX1a shared high similarity with other known alternative oxidases from different species. Four extrons and three introns were detected in the genomic DNA sequence, and the result of Southern blotting analysis suggested that NgAOX1a is a single copy gene.2. Northern blotting analysis showed that the transcript levels of NgAOX1a can be markedly accumulated when tobacco seedlings were treated with various abiotic stimuli, such as exogenous signaling molecules for plant defense response, salicylic acid (SA) and H₂O₂, and exogenous tricarboxylic acid (TCA) cycle metabolite citrate. However, it could be suppressed by abiotic stress, such as CoCl2, an inhibitor of ethylene (ET). In addition, NgAOX1a can also be strongly induced by three viral pathogens, Tobacco mosaic virus (TMV), Potato virus X (PVX) and Potato virus Y (PVY). Those results indicate that NgAOX1a may be involved in multi signal transduction pathways and may play an important role in defense response.3. Construct a sense expression vector pBI121-NgAOX1a, and transformed it into the tobacco plant (NC89). We also transformed the empty vector into tobacco at the same time as control. We carried out PCR identification and semi-quantitative RT-PCR analysis on some transgenic plants, and found that NgAOX1a has been expressed successfully in the tobacco plants.4. We analyzed the biological function of the T1 generation plants. On the basis of the molecular identification, the T1 transgenic plants heredity is consistent with the separate rule of 3:1.5. We obtained the promoter sequence of the NgAOX1 gene (GenBank Accession No. GQ199607) by LA PCR and reverse PCR. Using the analysis soft PlantCARE, we find that there are promoter core elements, such as TATA box and CAAT box, and a series of some putative cis-acting elements involved in SA responsiveness, light responsiveness, fungal elicitor responsiveness, and defense and stress responsiveness and so on.6. Construct six sense expression vectors for deletion ananlysis of the promoter, and transformed them into the Arabidopsis thaliana. And we have collected some transgenic seeds of the T0 generation.