Regeneration And Functional Characterization Of NtAOX1α-transgenic Tobacco

The mitochondrial respiratory chain in higher plants consists of the cyanide (CN)-sensitive cytochrome pathway and CN-resistant alternative pathway. The latter is catalysed by the alternative oxidase (AOX) at the surface of the mitochondrial inner membrane. AOX is encoded by a small family of nuclear genes. Expression of AOX genes and AOX protein is induced in response to diverse biotic and abiotic stress. It has been suggested that AOX plays a crucial role in maintaining homeostasis under varying growth conditions and in protecting plants against the lethal effects of reactive oxygen species (ROS). AOX expression is responsive to low temperature and has also been implicated in resistance to viruses. However, the issues of whether AOX affects ROS accumulation at low temperature and pathogen attack, and whether there exists additional function for AOX, have yet to be determined.In this research, AOX gene was isolated from Nicotiana tabacu Samsun (NtAOX1a). Two different plant expression vectors containing NtAOX1a cDNA in sense or antisense orientation were constructed and then introduced into Samsun by Agrobacterium tumefaciens-mediated transformation. A number of independent transgenic tobacco lines with altered AOX expression levels were obtained and homozygous T₃ plants were selected for functional analysis. The main results are as follows:1. The NtAOX1a coding sequence was obtained from NN-type Samsun tobacco by reverse transcription-PCR (RT-PCR) using primers based on the published AOX1a sequence from Bright Yellow tobacco. The full length of the cDNA is 1071bp.2. The cDNA was inserted in sense or antisense orientation under control of the 35S cauliflower mosaic virus promoter (CaMV35S) in the expression cassette of pBI121. Empty transformation vector control was also generated. The expression cassettes were introduced into Agrobacterium tumefaciens (strain LBA4404) and then used for tobacco leaf disk transformation,so we got three different kinds of transgenic tobacco plants.3. The kanamycin segregation analysis in the seeds of T0 generation, combined with the PCR assay of T₁ transgenic plants, proved that the heredity property of transgenic progenies is coincident with the 3:1 seperation ratio.4. The plants whose progenies are coincident with the 3:1 seperation ratio were selected. Northern blot and Western blot were performed to make sure that NtAOX1a has successfully expressed in the transgenic tobacco.5. The plants who displayed representative phenotypes of their transgenic lines were selected for characterization. Analysis of NtAOX1a expression in T₃ plants were carried out by Northern blot and Western bolt. The result showed that NtAOX1a expression was inherited stably from T₁ to T₃ generation. Respiratory characteristics of T₃ plants showed that alternation in the expression level of NtAOX1a affected the AP capacities.6. On the basis of the molecular identification,T₃ generation plants were selected for functional analysis. Under low temperature conditions, transgenic plants harboring sense NtAOX1a show more tolerant than that of the control and antisense line. When inoculated with Tobacco mosaic virus (TMV), the antisense line displayed better viral resistance relative to wild type and the sense line,suggesting the negative relationship between the expression level of NtAOX1a gene and the viral resistance. Measurement of H₂O₂ accumulation implied that ROS accumulation in the mitochondria is most likely to be the resistance-inducing signal under the control of AOX. Furthermore, we also found an important role for NtAOX1a in germination under H₂O₂-induced oxidative stress when the cytochrome pathway was inhibited.