Functional Analysis Of ZmABP9 In Response To Stresses And Preliminary Study In Gene Editing Through CRISPR/Cas9 System In Cotton

Cotton is one of the most widely planted crops in the world,and it is also the main source of cottonseed oil and fiber.However,abiotic stresses,such as soil salinity and drought,negatively affect growth,development and yield in cotton.ZmABP9,which encodes a bZIP transcription factor called ABA responsive element binding protein 9-ABP9,was screened by employing a yeast one-hybrid system for proteins specifically interacting with ABRE2 motif of catalasel(Cat1)gene in maize(Zea mays).ZmABP9 expression significantly improves tolerance in Arabidopsis to multiple abiotic stresses,but little is known about its role in cotton.In addition,it is difficult to edit the cotton genome using CRISPR/Cas9 system due to the complexity of the cotton genome.Therefore,in the study,the ZmABP9 was transferred into cotton to analyze funtions by Agrobacterium tumefaciens-mediated transformation.Meanwhile,we conducted a preliminary study of gene editing mediated by suing a CRISPR/Cas9 system.The researches are very meaningful to create new cotton varieties with greater tolerance to salt or drought stresses and to research the functional genomics of cotton by CRISPR/Cas9 technology.In the present research,the ZmABP9 gene was introduced into upland cotton(Gossypium hirsutum L.)cultivar R15 by Agrobacterium tumefaciens-mediated transformation,and 12 independent transgenic cotton lines were obtained.Two representative transgenic lines(L24 and L66)were selected for a series of stress treatment experiments.The results showed that cotton plants overexpression ABP9 have enhanced tolerance to salt and osmotic stresses.The transgenic plants developed better growth phenotype and better root systems in a greenhouse and higher germination,the longer seedling,reduced stomatal aperture and stomatal density in a growth chamber.Under salt and osmotic stresses,chlorophyll,proline and soluble sugar contents increased in transgenic cotton leaves and the malondialdehyde(MDA)content was lower than in R15.Especially in drought conditions,relative water content(RWC)and survival rate of transgenic cotton were higher than those of R15.ABP9 overexpression not only upregulated the transcription levels of stress-related genes,but also enhanced oxidative stress tolerance,reduced cellular levels of reactive oxygen species(ROS)through increasing activities and gene expression of antioxidative enzymes,and alleviated oxidative damage to cell under salt stress.The tissue staining proved this point.In addition,ABP9-expressing cotton was more sensitive to exogenous ABA at seed germination,root growth,stomatal aperture and stomatal density.Taking callus and one-week cotyledon leaves as material,a stable and effient protoplast preparation and 40%PEG4000 mediated target gene transformation and transient expression system in cotton were established.The suitable target sites were screened from cotton phytoene dehydrogenase(PDS)gene,which was selected as the target gene.The CRISPR/Cas9 system achived efficient genome editing in PDS through transient expression in cotton protoplast and Agrobacterium mediated genetic transformation.Conjointly,overexpression of ZmABP9 conferred enhanced tolerance to multiple abiotic stresses in cotton through possibly involving in ABA-dependent signal transduction pathway and regulation of ROS metabolism.We can also achived target site mutation directly in target gene through CRISPR/Cas9 system in cotton.All these results provide valuable resources and powerful tool for the development of new cotton varieties with resistance to abiotic stresses and research of functional genomics respectively.