Screening And Identification Of Anti-reverse Transcriptional Factor OoABF2 Interacting Proteins In Oxytropis Ochrocephala

In recent years,the degradation of grassland has become more and more serious,which has seriously affected the ecological balance and sustainable development.As one of the main poisonous grassland grass,O.ochrocephala has been widely spread due to its strong resistance and adaptability.The analysis of O.ochrocephala transcription data under three abiotic stresses(ABA,NaCl and PEG)showed that ABF2 transcription factors may be involved in the environmental resistance.Nevertheless,the mechanism of its detailed regulation process is still unclear.Therefore,based on the previous study of our group,this study intends to screen and identify upstream proteins of OoABF2 transcription factors to provide theoretical foundation for the molecular mechanism of resistance and expound the phenomenon of the spread of O.ochrocephala.(1)in this study,OoABF2 transcription factors(accession number:KU877410.1),were screened as the entry point,and SnRK2 s protein kinase family was selected as the target genes according to the previous research work and domestic and foreign literature reports.All eight SnRK2 s kinases from O.ochrocephala.were cloned and named as OoSnRK2.1-OoSnRK2.8(accession number: MH636330-MH636337).(2)using yeast two-hybrid technique,OoABF2-pGADT7 were ligated by T4 DNA ligase,and OoSnRK2s-pGBKT7 were ligated to construct eukaryotic recombinant plasmid.OoSnRK2.1 and OoSnRK2.6 were identified with the function of interacting with OoABF2 in yeast.(3)the recombinant plasmid of OoSnRK2.1-pSPYNE,OoSnRK2.6-pSPYNE and OoABF2-pSPYCE was constructed.The relationship between OoABF2 and OoSnRK2.1 / OoSnRK2.6 was verified in tobacco epidermal cell using bimolecular fluorescence complementary method.The interaction localization were in the nucleus.(4)OoSnRK2.1/OoSnRK2.6 were connected with pGEX-4T-1,OoABF2 and pET-32 a were ligated,and three soluble target proteins were obtained from E.coli.The interaction between OoABF2 transcription factor and OoSnRK2.1/OoSnRK2.6 was further confirmed by Pull-Down technique in vitro.(5)by various bioinformatics methods the basic physical and chemical properties of OoSnRK2.1 and OoSnRK2.6 were analyzed.The results showed that the target proteins were hydrophilic proteins with no transmembrane domain and signal peptide.The subcellular localization in nucleus,including specific conserved domains and phosphorylation sites.The phylogenetic tree stated that OoSnRK2.1 protein had the highest consistency with Tribulus terrestris(XP 003615157.1)and chickpea(XP 004490378.1).OoSnRK2.6 also had the highest homology with Tribulus terrestris(XP 013456889.1).(6)the expression profiles of OoSnRK2.1 and OoSnRK2.6 were analyzed by qRT-PCR under three abiotic stresses(ABA,NaCl and PEG).The data demonstrated that three genes could be activated by ABA.At the same time,the transcription level reached a higher level at 3 h.Under the treatment of NaCl and PEG,the expression of OoSnRK2.1 and OoSnRK2.6 was also induced.The results suggested that target genes can resist the adverse environment relying on the ABA signaling pathway.In conclusion,this study suggests that OoABF2 and their upstream OoSnRK2.1/ OoSnRK2.6 kinases dependent on ABA signaling pathway play an important role when faced with adverse environment,which is significative in revealing and elucidating the mechanism of stress resistance.