Cloning And Functional Analysis Of Arabidopsis Thaliana ANK-TM2 Gene Family

In nature,plants are often affected by various environmental factors such as drought,water logging,salinity,high and low temperature,etc.These stress factors will have a series of effects on the growth,development and metabolism of plants,and may even cause plant death in serious cases.Therefore,it is an important direction in the field of plant research to clarify the molecular mechanism of plant resistance to adverse environmental factors and to improve the ability of resistance to environmental stress.This thesis focuses on the priliminary study of ANK-TM2 unknown function gene family of Arabidopsis thaliana,and the exploration of effects these genes may have during the process of plants'stress response.Firstly,T-DNA insert mutant seeds were purchased from NASC and identified to obtain homozygous mutant plants.After that,these mutant plants were compared with wild Arabidopsis in respect of their phenotypic difference under various stress conditions,so that the effects of these genes on plants'different stress responses were detected.After the preliminary results were obtained,expression vectors of candidate genes were then analyzed and constructed through molecular biology technique.Finally,by using the agrobacterium infection method to transfect Arabidopsis thaliana,homozygous expression vector plants were screened,followed by subsequent studies on functions and mechanisms of action of these genes.The main research results were as follows:?1?The acquisition and screening of T-DNA insertion mutant of each gene from the ANK-TM2 gene family:Through searching for T-DNA insertion mutant of related RT-qPCR)was used to detect the expression of target genes in mutants and wild-type Arabidopsis thaliana,and conclusion was made that the gene expression of ank-tm203and ank-tm204 was significantly lower than that of wild-type Col-0,while the expression of ank-tm207 and ank-tm208 was obviously higher than that of wild-type Col-0.Considering that different T-DNA insertion sites will lead to different expression changes,we believed that the expression of these genes had changed,and could be used for the next physiological phenotypic studies on stress responses.?3?ANK-TM2 homozygous mutants have phenotypes under different abiotic stress:The homozygous mutants were treated with drought,high salt,low calcium and high calcium,etc.The effects of target genes on plants'stress response were analyzed by observing their phenotypic differences with wild plants under different treatments.The results showed that the root length of ank-tm204 was shorter than that of wild type plant under 0mM CaCl₂ condition,while the root length of ank-tm208was longer than that of wild type plant under 0mM CaCl₂ condition.The root length of ank-tm207 was shorter than that of wild type plants under conditions of high calcium,high salt and drought,etc.It could be seen that ANK-TM204,ANK-TM208and ANK-TM207 genes might play an important role in plant responses to external calcium stress,salt stress and drought stress.?4?ANK-TM2 homozygous mutants have phenotypes under different biotic stress:The wild type and homozygous mutant of Arabidopsis thaliana were injected with PsmES4326,and after two to three days,the disease resistance of Arabidopsis thaliana was determined by comparing the amount of the bacterial plaque in leaf.Results showed that ank-tm202,ank-tm204,ank-tm207 and ank-tm208 showed obvious resistance to disease.?5?For further research,we have constructed plant expression vectors by adopting Gateway technique:GFP fusion expression vector,over-expression vector,and GUS fusion expression vector of ANK-TM204,ANK-TM207 and ANK-TM208were constructed on the basis of pGWB406,pGWB502?and pGWB533 expression vectors.The next step was that by using the agrobacterium infection method,the Arabidopsis thaliana was transformed,and the homozygous expression vector plants were screened,followed by subsequent studies on the functions and mechanisms of action of these genes.