Screen Of Arabidopsis Transcription Factors In CO₂ Signaling And Heat Stress By Artificial Microrna Interference Technology

Artificial miRNA interference is an efficient gene silencing technology which is widely used in the studies of gene function.AmiRNA are generated based on the endogenous miRNA precursors by replacing the endogenous miRNA with the artificial designed amiRNA sequence.It is widely used in studies of overlapping function genes.Traditional forward genetic screens and reverse genetic screens based on T-DNA insertion using diverse mutagens are limited in identification of functional redundant genes.In the plant,there is a huge number of transcription factors in which most of them are functional overlapping redundant genes,thus it is really difficult to study them.To date,there isn't any transcription factor identified to function in CO2-induced stomatal movement.And most of transcription factors identified in heat stress are functional redundancy.To solve this problem,this study generated a transcription factor mutant library by using artificial microRNA interfere technology and proper methods were used to screen some responsive to CO2 signaling and heat stress mutants.The main results of this study are as follows:1.We generated an arabidopsis amiRNA interference mutant library targeted to transcription factors.The amiRNA transcription factor mutants group was generated using Arabidopsis ecotype Col-0 as the background plants.In this study,website WMD3(Web mirnas Designer)was used to design the amiRNAs whose target genes are predicted to be transcription factors in Arabidopsis Thaliana.Anyone of the amiRNAs was designed to target at least two members of its gene family.After vectors construction,the amiRNA library pool was transformed to the Col-0 plants by agrobacterium-mediated transformation using flower dipping method,we successfully obtained many transgenic plants containing different amiRNAs.2.Some mutants impaired in CO2-induced stomatal movement were obtainedTo screen the mutants impaired in CO2-induced stomatal movement,we compared the leaf temperature of different plants before CO2 treatment(400ppm)and after treatment(800ppm).By this method,we obtained twelve independent mutants that were insensitive to high CO2 treatment.Further studies on ciml(CO2-insensitive mutant 1)mutant showed that ciml was insensitive to CO2.It is suggested that mutants obtained are impaired in CO2-induced stomatal closure.3.Some mutants related to heat stress were obtainedTo get the heat stress mutants,we modified the screening method based on the method previous reported,the heat stress screening conditions were determined:38 ?processing 1.5 hours,21 ? recovering for 2 hours,50 ? treating for 2 hour.After the first screening,several mutants were obtained for further studies.The mutants provided the experimental materials for subsequent research.