Studies Of Ethylene On The Mechanism Of Silicon-enhancement Of Salt Tolerance In Tobacco BY-2 Cultured Cells And Preparation Of Alternative Oxidase Polyclonal Antibody

In the present study, the tobacco cultured cells (BY-2) and wild-type Arabidopsis thaliana (WT) were used to the experimental materials. Firstly, the physiological responses of tobacco BY-2 in a long-term salt stress and the relief of silicon to salt stress were studied. And the relationship between ethylene and silicon actin under salt stress the influence of silicon on ethylene content were further investigated. The possible physiological function and mechanism of ethylene in silicon enhanced tolerance to BY-2 cell under salt stress were disccussed.Secondly, AOX polyclonal antibody of Arabidopsis was successfully produced. The main results were summarized as follows:1. The cell death rate of BY-2 cell at the lower salt (50 mM NaCl) treatment was almost no change. The cell death rate of BY-2 cell at 100 mM and 200 mM treatments increased to 32% and 68%, respectively. The cell vitality of BY-2 cells both in low and high salt concentrations markedly decreased and MDA contents obviously increased, suggesting that the integrity of the cell membrane was oxidative damage by salt stress. The damage levels of all physiological index tested in 100 mM NaCl treatment were moderate, therefore 100 mM NaCl was used to further study.2. The exogenous silicon could significantly relieve the impact of salt stress on cell morphology and reduce the cell death rate under salt stress. The decrease of growth and vitality of BY-2 cells in salt stress were partially recovered by the exogenous silicon. The increase of MDA content was also markedly reduced by silicon.3. It was found that the relief action of silicon to BY-2 cells in salt stress need the involment of ethylene by the treaments of AgNO3 (an ethylene receptor inhibitor) and AOA (an ethylene synthetic inhibitor). The protection of silicon was not observed and the more damage appeared when the role of ethylene was not worked.4. The release of ethylene in BY-2 cells increased with the time of silicon treatment increase. It increased to 1.5 times of the control in the silicon treatment for 48 h. The expression of ACS and ACO which are the key enzyme genes of ethylene synthesis were increased in silicon treatment. It was illustrated that the enhancement of ethylene content in silicon treament was due to improve the genes expression of ethylene synthesis enzymes.5. The content of reactive oxygen species (ROS) in BY-2 cells at the silicon treatment was almost no effect. But the ROS content in BY-2 cells resulted in marked increase at Si+AgNO3 treament. The ROS contents in BY-2 cells at Si+AgNO3 treatment for 24 h and 48 h increased by 1.4 and 2.0 times, respectivrely. The balance between ROS and the defense system was broken and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in BY-2 cells greatly changed by Si+AgNO3 treament. POD activity in BY-2 cells at silicon treatment increased which enhanced more in Si+AgNO3 treatment. CAT activity was no difference in silicaon treatment and decreased in Si+AgNO3 treatment. SOD activity increased in silicon treatment and declined in Si+AgNO3 treatment.6. Alternative oxidase (AOX) is the terminal oxidase in the cyanide-resistant respiration pathway in plant mitochondria. In order to further study on AOX function, the coding region of AOX gene in Arabidopsis thaliana was sub-cloned into the prokaryotic expression vector pET-28a, the recombinant vector was introduced into E.coli BL21 (DE3) cells for efficient expression. The soluble His-fusion protein was purified through His-column and used to immunize rabbit, finally AOX polyclonal antibody was successfully produced.The specificity and titer of polyclonal antibody were also identified.The polyconal antibody of rabbit AOX will facilitate further funcitional study of AOX.