Gene Cloning And Functional Identification Of FtbZIP5 Resistance-related Transcription Factor From Tartary Buckwheat

Tartary buckwheat（Fagopyrum tataricum）is a kind of characteristic small coarse grain rich in rutin and other flavonoids,which has excellent anti-stress production performance.In recent years,the research on stress resistance mechanism of Tartary buckwheat mainly involves stress physiological and biochemical analysis and the mining of stress-related transcription factors.As one of the largest transcription factor families in plants,bZIP transcription factors are widely involved in various stress response processes of plants.AREB/ABF transcription factors of A sub-family can mainly participate in the response of plants to osmotic stress through ABA dependent signaling pathway.Studies have shown that the SnRK2-AREB/ABF regulatory system participates in plant responses to drought and salt stress,and acts as a central hub in plant response to abiotic stress,participates in signal regulation networks.In this study,we screened the transcription factor gene FtbZIP5,which can respond to drought/high salt/ABA treatment rapidly,and cloned and analyzed its cDNA and promoter sequence;to identify the subcellular localization,transcriptional activation activity and specific binding sequence of its coding protein;to identify the biological effect of FtbZIP5 by the technology of Arabidopsis transgene;the yeast two-hybrid technology was used to screen the potential phosphorylation and functional regulation of FtSnRK2s in the interaction of SnRK2-AREB/ABF were identified.Additionally,the significance of FtbZIP5in ABA signaling pathway was discussed by using transgenic technology to supplement Arabidopsis mutant abi5-1（abscisic acid-insensitive 5-1）.This study preliminarily elucidated the molecular mechanism of Tartary buckwheat transcription factor FtbZIP5 involved in plant resistance to external environmental stress,which not only helps to deepen the understanding of Tartary buckwheat’s excellent resistance performance,but also provides reference for Tartary buckwheat’s resistance molecular assisted breeding.The main results are as follows:1.Molecular characterization of Tartary buckwheat transcription factor FtbZIP5.After the treatment of drought,high salt and ABA,the expression of FtbZIP5 gene was significantly increased（P<0.05）,indicating that FtbZIP5 may be involved in the stress response of Tartary buckwheat.Based on the transcriptome data of Fagopyrum tataricum,the cDNA of FtbZIP5gene was cloned.The sequence analysis showed that the ORF was composed of 419 amino acids with a molecular weight of 45.671 kDa and an isoelectric point of 8.46.The results of the multiple sequence alignment analysis show that FtbZIP5 has not only a bZIP domain but also potential phosphorylation peptides,C1,C2,C3 and C4,similar to other identifified AREB/ABFs.Phylogenetic analysis showed that FtbZIP5 was closely related to AREB/ABF transcription factors involved in osmotic stress.The pBridge-FtbZIP5 vector and subcellular localization vector were constructed and transferred into AH109 yeast cell and Arabidopsis respectively.The results showed that FtbZIP5 had transcriptional activation activity and located in the nucleus.Yeast one-hybrid and tobacco leaf co-transfection experiments showed that FtbZIP5 can interact with ABRE elements to enhance the expression of downstream reporter genes.2.Cloning and bioinformatics analysis of the promoter of Tartary buckwheat transcription factor FtbZIP5.Based on the genomic data of Tartary buckwheat,the 2066bp sequence upstream of FtbZIP5 gene was cloned and designed P_FtbZIP5.The analysis of online website Plant CARE shows that P_FtbZIP5contains ABA response element（ABRE）,methyl jasmonate response element（TGACG-motif）,defense stress response element（TC-rich repeats）and low temperature response element（LTR）which are mainly involved in stress signal response.3.The effect of Tartary buckwheat transcription factor FtbZIP5 on the stress resistance of transgenic Arabidopsis.The plant expression vector pCHF-FtbZIP5-YFP was constructed and the transgenic Arabidopsis plants were obtained by Agrobacterium tumefaciens.Seed germination and root length experiments showed that overexpression of FtbZIP5 significantly increased the resistance to salt and drought stress and increased the sensitivity to ABA in germination and seedling stages.Under drought and salt stress,compared with WT,the activity of antioxidant enzymes（POD、SOD and CAT）and proline content of FtbZIP5 were significantly higher than WT（P<0.05）,while MDA content was significantly lower（P<0.05）,and ROS accumulation decreased.Environmental stress and expression of ABA responsive genes showed that the gene expression levels of RD29A、RD29B、RAB18、RD26、RD20 and COR15 in the transgenic lines was significantly higher than that in the wild type（P<0.05）.4.Effect of overexpression of Tartary Buckwheat FtbZIP5 on ABA sensitivity of Arabidopsis mutant abi5-1.The plant expression vector pCAMBIA1305-FtbZIP5 was constructed,and the Arabidopsis mutant abi5-1 was transfected by the flower soaking method and the transgenic positive strain was obtained.The results of restorer experiments showed that overexpression of FtbZIP5 could partially complement the sensitivity of Arabidopsis seeds to exogenous ABA during germination.5.Screening of FtSnRK2s protein interacting with FtbZIP5.Based on the analysis of protein interaction of transcriptome data,11 protein kinase sequences of SnRK2s family were screened.The phylogenetic tree was constructed with SnRK2s protein family of Arabidopsis,and three protein sequences that may interact with FtbZIP5 were predicted.The results of yeast two hybrid experiment showed that there was physical interaction between FtbZIP5 and FtSnRK2.6,but not directly with FtSnRK2.2/2.3.The above results indicate that FtbZIP5 of Tartary buckwheat increases the transcription level of antioxidant-related genes and antioxidant enzyme activity and reduces ROS accumulation through the ABA-dependent signaling pathway under the regulation of protein kinase FtSnRK2.6,thereby increasing the stress tolerance of plants.