Studies On Molecular Mechanism Of AtpA Gene Regulating Plants' Response To Low Temperature Stress In Arabidopsis

Low temperature is one of the main environmental factors that limit the normal growth of plants.In order to resist low temperature disasters,plants have evolved their own unique resistance mechanisms.When suffering from low temperature,plants can rapidly initiate the expression of downstream genes by sensing changes in cell membranes,the flow of calcium ions or other receptors to enhance their cold resistance.The function research of stress-related genes is very important to understand the stress resistance mechanism of plants under stress and improve the plant's stress resistance.In the early stage of this laboratory,a strain of Gliocladium pink was isolated from the soil.In the study of the fungus triggering tomato defense response,it was found that it and the pathogenic bacteria together induced a key gene ATP synthase CF1?subunit?atpA?Gene is up-regulated,and atpA gene also plays an important role in tomato response to low temperature stress.Atp A is a photosynthesis-related chloroplast gene that participates in energy metabolism and is relatively conservative in evolution.Studying the role and mechanism of the model plant Arabidopsis atpA gene under low-temperature stress can help provide a theoretical basis for stress tolerance of other crops.This study intends to use molecular biology,biochemistry,genetics and other methods to systematically study the role of atpA under low temperature stress and to explore its mechanism in depth.These studies will help elucidate the mechanism of plants responding to low temperature stress,provide theoretical basis for guiding crop improvement and molecular breeding,and provide important guidance methods for the rapid and healthy development of China's agricultural economy.The results of this study are as follows:?1?The Arabidopsis atpA gene cloned in this study is a chloroplast gene.It encodes the ATP synthase CF1?subunit protein.The expression pattern of the atpA gene was explored through q RT-PCR experiments.It was found that the atpA gene was expressed in various tissues in Arabidopsis except the roots,and the expression level in the pods highest.?2?To investigate whether the expression of atpA gene is related to cold stress through fluorescent quantitative PCR experiment.The experimental results show that the content of atpA gene will change with the change of low temperature treatment time,indicating that atpA gene is induced by cold stress.And through the Arabidopsis protoplast transient transfection technology to explore the Arabidopsis atpA gene subcellular localization.It is concluded that the protein encoded by the atpA gene is located on the chloroplast.?3?Atp A mutants treated at 4°C had the same phenotype as wild-type Arabidopsis.DAB staining found that atpA mutants had no difference with wild-type Arabidopsis.However,the results of freezing injury experiments showed that the Arabidopsis atpA mutants had higher survival rate than wild-type Arabidopsis after freezing injury,indicating that the atpA gene negatively regulates Arabidopsis cold resistance.?4?Observation and determination of the photosynthetic system damage of Arabidopsis atpA mutants after cold stress with a plant fluorescence imager.The results indicate that the photosynthetic system damage of atpA mutants is weaker than that of wild type.Through physiological and biochemical experiments,it was found that after cold stress,the enzyme activity of the chloroplast complex?F1F0-ATP synthase?has decreased,but the decline of wild-type Arabidopsis is greater than that of atpA mutants.After cold treatment,the ATP content will increase,But the increase of the atpA mutant is greater than that of wild-type Arabidopsis.?5?Through fluorescence quantitative PCR experiments,it was found that atpA gene will affect the expression of CBFs gene.In addition,ROS-related genes?CAT,FER1,RBoh D,APX1?are highly expressed in atpA mutants.?6?Determined by non-damage micro-testing technology?NMT?,the results show that after cold stress,the rise of atpA mutant Ca²⁺outflow is slightly higher than that of wild type,and the increase of H⁺outflow signal is much larger than that of wild type H⁺outflow The amplitude of the signal rise.?7?In order to explore the potential interaction proteins and interaction positions of atpA,after bioinformatics analysis,yeast two-hybrid experiment,pull down and bimolecular fluorescence complementary experiment,the results show that atpA can directly interact with OST1.And the action position is on the chloroplast.?8?Experiment with isotope labeling.The results show that OST1 cannot phosphorylate atpA.The research will lay a theoretical foundation for further elucidating the mechanism of plants responding to low temperature stress,and at the same time provide theoretical basis and genetic material for guiding crop improvement and molecular breeding.