The Role And Its Mechanism Of ANAC055 For Drought Stress Response In Arabidopsis Thaliana

Drought has huge impact on plant growth,development and economic yield,and could even lead to plant death.Therefore,it is a prime scientific issue to study the genetic basis and molecular mechanism of plant drought tolerance and to explore an effective way for drought resistant plant breeding.In this study,to identify the important role of ANAC055 in drought stress response in Arabidopsis thaliana,we produced ANAC055 knockout plants using CRISPR/Cas9 technology and investigated phenotypes of these knockouts under drought stress based on analysis of the ANAC055 phylogeny and the influence of drought stress on ANAC055 expression in Arabidopsis thaliana.Downstream target genes and interacted proteins of ANAC055 were further studied by yeast two-hybrid and BiFC and other techniques to reveal the molecular mechanism of ANAC055 regulating drought stress response.Our main results are as following:1 The ANAC055 gene belongs to NAC transcription factor family and islocated on chromosome 3 of Arabidopsis thaliana.Its encoded protein(ANAC055)contains a conserved NAM domain,and is localized to the nucleus in Arabidopsis thaliana.2 Expression of ANAC055 is induced by drought stress.A highest expression level of ANAC055,up to 13-fold increase of the control plant was induced 3 hours after drought stress.3 Two homozygous ANAC055 knockout mutant materials were obtained using CRISPR/Cas9 technology.Under drought stress,the aboveground fresh weight of the two mutant materials showed no significant difference from that of the WT,but the length of roots reduced markedly than those of the WT.Significantly higher leaf water loss rate in ANAC055 knockout mutants compared to the WTs were also detected,showing that the ANAC055 knockout mutants were more sensitive to drought stress.4 ZF2,AT2G31945,MYB2 and ERD1 were indentified to be interacted with ANAC055 in yeast two-hybrid and BiFC analysis.They are mainly localized to the nucleus.5 Four knockout vectors of ZF2,AT2G31945,MYB2 and ERD1 genes were successfully constructed.Functional homozygous knockout mutants of ZF2 and heterozygous knockout mutant of MYB2 were obtained.Further generation of homozygous tera knockout of these four gene and identification of their drought-resistant phenotypes are yet to be required.In conclusion,our results preliminarily indicated a potentially important role of ANAC055 for drought stress response in Arabidopsis thaliana.Whether it interacts with ZF2,AT2G31945,MYB2,ERD1 and other proteins to jointly regulate drought stress response requires further study...