Cloning And Functional Analysis Of Tomato Transcription Factors Related To Alkali Stress

As the main abiotic stress,saline-alkali stress is an irreversible obstacle for the growth and development of plants which can affect crop yield and quality.Compared with salt stress,the molecular mechanism of alkali stress is more complicated.Under alkaline stress,the plant's early response is similar to salt stress,and the gene regulatory network involved during this period is faster and more complex than that of salt stress.Tomato,as an important cash crop and model crop,is of great significance for studying the molecular mechanism of plants response to alkali stress.In our previous experiments,we performed high-throughput transcriptome sequencing with the root tips of Arabidopsis thaliana under alkaline stress,found transcription factors significantly induced by alkali stress,constructed tomato overexpression and mutant transgenic plants,and explore the molecular mechanism of tomato tolerance against alkali stress,the specific results are as follows:?1?Through cytological observation of Arabidopsis thaliana,we found that alkali stress inhibited the cell division activity of the meristematic zone,and inhibited the size of the meristematic zone and elongation zone to inhibit root elongation.?2?Alkali stress promoted the expression of auxin reporter DR5:GUS and the expression of auxin synthesis related genes,indicating that alkali stress may affect root growth by regulating the auxin synthesis in root tips.?3?We detected the content of H₂O₂ and O₂^-by DAB and NBT staining respectively under alkali stress,and found that H₂O₂ and O₂^-were significantly higher.Simultaneously,ROS-related genes were significantly affected by alkali stress.These results indicate that alkali stress promotes the accumulation of ROS in the root tips.?4?Alkali stress caused root cell death and induced the expression of genes related to DNA damage repair,indicating that alkali stress can cause DNA damage and repair response in root tips.?5?Transcriptome analysis was performed on root tips treated with alkali stress.13,539differentially expressed genes were identified,of which 6,634 were up-regulated and 6,093were down-regulated.Analysis of differential expressed genes at various time points showed that there were as many as 8539 differentially expressed genes were found at 48 h after alkali stress treatment,and 118 genes were significantly regulated at all time points.According to the GO analysis,it was found that the salt stress signaling pathway,ABA signaling pathway,ion stress pathway,osmotic stress pathway,hormone synthesis pathway,redox signaling pathway and other pathways were involved in the response process of alkali stress in plants,indicating that the root response to alkali stress is a complicated process.?6?Transcriptome analysis showed that 1012 transcription factors were significantly regulated by alkali stress.Among them,MYB,bHLH,NAC,ERF and WRKY transcription factor families account for the majority.We further selected 28 transcription factors significantly regulated by alkali stress and found tomato homologous genes.We performed qRT-PCR in tomato to verify their homologous transcription factors were also significantly regulated in tomato under alkaline stress,indicating that these 28 transcription factors may play important roles in tomato response to alkali stress.?7?In order to explore the role of the above tomato transcription factors under alkali stress,we constructed overexpression vectors and genome editing?CRISPR/Cas9?vectors of the 28 tomato transcription factors,and we have obtained T0 plants of 2 overexpression vectors,and T0 plants of 1 genome editing?CRISPR/Cas9?vector.