The Expression Analysis And The Functional Identification Of OsAOX1Gene Family Under Stress Condition

The mitochondrial respiratory chain in higher plants consists of two pathways, the cyanide (CN)-sensitive cytochrome pathway and CN-resistant alternative pathway. The latter is catalyzed by the alternative oxidase (AOX) at the surface of the mitoch-ondrial inner membrane. The alternative pathway branches electrons at the ubiquinone pool of the cytochrome pathway and reduces oxygen to water without conservation of energy in the form of ATP, energy is emited as heat. The alternative oxidase is a member of the binuclear iron carboxyl proteins family. The alternative oxidase is encoded by a small family of nuclear genes, and expression of AOX genes is induced by diverse biotic and abiotic stress. The cyanide-resistant alternative pathway and the biological function of AOX are always the hot spot of plant physiological research. Now studies of AOX are more concentrated in dicots than monocots. Previous studies focus on the function of the whole AOX in single stress, such as cold and drought. But the function studies of the memebers of AOX are very little, and it is still an open question for the signaling pathway of AOX.In our research, according the reported sequences, we designed the primer pairs for the AOX gene and studied its expression pattern of it under different inducible conditions, such as cold, hot, salanity, drought, and peroxide drugs. We cloned AOX1α and AOX1b genes from rice and conducted a series of studies have on the evolution and sequence analysis. Different expression vectors were constructed and then introduced into rice by Agrobacterium tumefaciens-mediated transformation. A number of independent transgenic homozygous T2lines were generated and selected for functional analysis. Meanwhile, we analyzed physiological indicators of overexpression plants in adverse situation, such as ion leakage rate and MDA content. The main results are as follows:(1) According to the bioinformatics analysis data, we designed OsAOX1α, OsAOX1b and OsAOX1c (OsAOX1α/b/c) specific primers and analyzed the expression pattern of them. We found that the expression of OsAOX1α and OsAOX1b was induced under all stress treatments, whereas the expression of OsAOX1c was not obviously altered following any treatment. The induction of OsAOX1α and OsAOX1b expression responded to cold temperature, drought, and salinity in a time dependent manner. The highest induction level were achieved at24h of drought, cold temperature and salinity treatment, with12.3-,17.5-and30.7-fold increases in OsAOX1α expression level and15.2-,7.1-and36.6-fold increases in OsAOX1b expression relative to the untreated control, respectively. The induction of OsAOX1α expression by the hot-temperature stress was also time dependent manner; the highest induction level (an8.9-fold increase in expression) was observed after24h of treatment. The peak of hot temper-ature induced OsAOX1b expression was observed at1h with a10.6-fold increase in expression. The MV-mediated induction produced a38.8-fold increase in OsAOX1α and a117.2-fold increase in OsAOXlb expression following the24h treatment, where-as fluctuating induction levels were observed with the H2O2treatment, resulting in no more than a five-fold increase in the expression of either gene.(2) OsAOX1α and OsAOX1b genes were cloned from rice by reverse transcription-PCR (RT-PCR). The full-length CDS of OsAOX1α was999bp, including four extrons and three introns. The gene prediction encodes333amino acids, with molecular weight of37136.5D and isoelectric point of8.2369. The full-length CDS of OsAOX1b was1008bp, including three extrons and two introns. The gene encodes336amino acids, with molecular weight of37250.5D and isoelectric point of8.04.(3)We constructed OsAOX1α and OsAOX1b overexpression vectors, and named OE-OsAOX1α and OE-OsAOX1b. Then we transformed vectors into rice by Agrobac terium tumefaciens-mediated infection, so we got transgenic rice plants.The hygromycin segregation analysis in the seeds of To generation, the plants whose progenies are coincident with the3:1seperationratio were selected.Combined with the PCR and western blot assay of T1transgenic plants to prove that OsAOXla and OsAOXlb has successfully expressed in the transgenic rice.(4) The T1plants displaying representative phenotypes of their transgenic lines were selected for T2generation。Analysis of OsAOX1α/b expression in T2plants were carried out by Western bolt and PCR. The result showed that OsAOX1α/b expression was inherited stably in T2generation. On this basis, the T2generation plants were selected for functional analysis. Under low temperature stress, the cold-resistant of the overexpression plants improved significantly relative to the control. Compared to control plants, overexpressing transgenic plants show greater cold resistant, either the aerial parts or the underground parts. The root length and shoot length of plants under cold stress, were3.8-fold and3.2-fold increase in OE-OsAOX1a linel transgenic plants and2.8-fold and2.5-fold increase in OE-OsAOXla linel transgenic plants relative to the treated control, respectively. The root length and shoot length of plants under cold stress, were5.2-fold and5.8-fold increase in OE-OsAOX1b line3transg-enic plants and4.8-fold and6.0fold increase in OE-OsAOX1b line4transgenic plants relative to the treated control, respectively.(5) On the basis of function analysis, we measured physiological indicators of overexpression T2plants in drought and cold temperature, such as ion leakage rate and MDA content. The ion leakage rate of cold temperature, with1.1-fold and1.22-fold decrease in two different transgenic plants of OE-OsAOX1a and1.45-fold and1.57-fold decrease in two different transgenic plants of OE-OsAOX1b relative to the control, respectively. The MDA content of cold stress, with1.1-fold decrease in linel of OE-OsAOXla transgenic plants and1.32-fold decrease in line2of OE-OsAOXla transgenic plants; and1.30-fold in line3of OE-OsAOX1b transgenic plants and1.35-fold in line4of OE-OsAOX1b transgenic plants. The results show that the overexp-ression OsAOX1α and OsAOX1b make the damage levels of cell membrane is reduced, and this mechanism is likely regulated by reactive oxygen species dominated signal-ing pathway.