The Functional Analysis Of AOX In Arabidopsis Thaliana

Alternative oxidase is the terminal oxidase of the alternate oxidase pathway in the respiratory chain of plant mitochondria.It is mainly responsible for transferring electrons from UQ pool to O₂ to form H₂O in the plant mitochondrial electron transport chain.It is also an important sensor of cell redox balance,which helps maintain the dynamic balance of metabolism in mitochondria.In the process of plant growth,it is inevitable to suffer stress from the external environment,such as high temperature,cold injury,high light,heavy metal,salt,drought and so on,thus growth pattern will change,so plants develop an internal defense mechanism to sense subtle changes in the external environment and thus respond to stimuli given by the external environment.The existence of AOX in higher plants provides a great role for the growth and development of plants.Not only AOX can transfer electrons,but also remove the reactive oxygen species produced in plants.At present,there are few studies on aox mutants,so this experiment mainly uses aox mutants as materials to study the functional mechanism of AOX,so as to increase the understanding of the functional characteristics and mitochondrial respiration of AOX.The AOX gene family consists of five genes:AOX1a?AOX1b?AOX1c?AOX1d and AOX2,they are encoded respectively by AT3g22370,AT3g22360,AT3g27620,AT1g32350 and AT5g64210.In order to study the function of AOX,the first step is to get mutants of different genotypes.Therefore,the main research contents of this experiment are as follows:1.Obtained homozygous single mutants of Arabidopsis thaliana and obtained aox1b mutant through CRISPR-Cas9 system;2.Other mutants with different genotypes were obtained by hybridization and CRISPR on the basis of the original mutants;3.To research the role of AOX gene in plant mitochondrial electron transport chain;4.Phenotypic analysis of mutants with different genotypes.In this experiment,two different line aox1b mutants were firstly obtained by CRISPR-Cas9technology,the result of Cas9 cleavage showed that one of the mutants had base addition and Cas9 was excised on the third gRNA,161 bp fragments were missing between the third gRNA and the fourth gRNA,as a result,the genome length was 160 bp shorter than wild type.The other line of the mutants had base addition,17 bases were missing on the fourth gRNA,a137 bp repetitive sequence was inserted before the fourth gRNA,as a result,the genome length was 121 bp longer than wild type.The protein primary structure prediction also indicated that the protein of two mutants had been destroyed.Secondly,based on the original four mutants aox1a,aox1c,aox1d and aox2,I obtained double mutant,triple mutant and quadruple mutant of different genotypes by hybridization and then quintuple mutant were obtained through CRISPR technology.At present,the identification of quintuple mutant is still in progress.Finally,the phenotypes of these mutants were observed and analyzed under salt stress and drought stress.The results showed that drought stress had little effect on mutants,but the mutant is sensitive to salt stress,salt treatment can affect plant growth status to some extent,and the content of hydrogen peroxide in the mutants,especially the double mutant and quadruple mutant,increased obviously after salt treatment,while other indexes were not significantly affected.However,the phenotypic analysis of mutants has only been done in part,and some treatment conditions are not very reasonable.This part still needs to be further studied.The experiment of blocking cytochrome pathway by antimycin A showed that AOX1a played major role in AOX pathway,and there may be functional redundancy in AOX gene.The acquisition of various mutant materials in this experiment and the current research on stress provide convenience for the subsequent experiments,which is conducive to further understanding of the functional mechanism of AOX in the future.