杨树转录因子PdbCRF5调控盐胁迫的分子机理研究

[Objective]Clarifying the function and regulatory mechanism of the PdbCRF5 gene in participating in the salt stress process of Populus davidiana×P.bolleana provides theoretical basis and genetic resources for improving plant salt tolerance through tree genetic engineering breeding.[Method]Early analysis of the salt stress transcriptome of Populus davidiana×P.bolleana found that the expression of PdbCRF5 gene was significantly induced by salt stress,indicating that it may participate in the regulation of salt stress of Populus davidiana×P.bolleana.By cultivating transgenic poplar plants that over-express and inhibit the expression of the PdbCR F5 gene,we found that the PdbCR F5 gene negatively regulates plant tolerance to salt stress.Subsequently,we conducted further analysis and identification of downstream genes and their interacting proteins regulated by PdbCRF5.[Result]1)The PdbCRF5 protein is located in the nucleus.Its expression is induced by high salt,drought,and ABA stress.2)Over-expression of PdbCRF5 gene negatively regulates salt tolerance in plants.Further research has found that inhibiting the expression of PdbCRF5 gene can improve plant salt tolerance by reducing the accumulation of reactive oxygen species and sodium ions in plant cells under salt stress.3)The ability of PdbCRF5 transcription factor to bind GCC(GCCGCC)and DRE(ACCGAC)elements was demonstrated through yeast one-hybrid and Agrobacterium tumefaciens transient transformation experiments in tobacco.4)Perform RNA-Seq analysis on over-expressed and wild-type Populus davidiana×P.bolleana.Compared with the control group,there were 166 up-regulated genes and 41 down-regulated genes in the over-expression group;By analyzing and searching the differential gene promoters,11 differential genes containing GCC elements and 27 differential genes containing DRE elements were found in the promoters,and most of these genes were reported to be involved in plant abiotic stress response.The results of the ChIP experiment showed that the PdbCRF5 transcription factor can bind to sequences containing GCC or DRE in the promoter fragments of 13 genes such as PdbHSP90-6.The qRT-PCR results showed that the expression of PdbHSP90-6 was significantly induced in transgenic plants after salt stress,indicating that it might play a more important role in the regulation of salt stress by PdbCRF5.5)Based on the previous salt stress RNA-seq data of Populus davidiana×P.bolleana,a salt stress regulatory network that PdbCRF5 gene may participate in was constructed through GGM algorithm.The results showed that there were 18 target genes might directly regulated by PdbCRF5.Furthermore,ChIP-PCR was used to validate the possible involvement of PdbCRF5 in salt stress regulation networks.We randomly selected 8 possible downstream target genes for validation,and the results showed that PdbCRF5 can bind to the promoter fragments of these 8 downstream target genes to varying degrees,indicating that the regulatory network is basically accurate and can be used for subsequent research.6)The transcriptional activation experiment found that the PdbCRF5 transcription factor has transcriptional activation activity.The yeast two-hybrid experiment demonstrated that the transcription factor PdbCRF5 of Populus davidiana×P.bolleana can interact with homologous proteins PdbCRF1,2,and 6 to form homologous dimers.In addition,over-expression of the PdbCRF6 gene also reduced the salt tolerance of transgenic plants.[Conclusion]Our results demonstrated that the PdbCRF5 transcription factor can participate in regulating salt tolerance in plants.Over-expression of PdbCRF5 reduces salt tolerance in transgenic plants,while inhibition enhances salt tolerance in transgenic plants.PdbCRF5 can participate in plant salt stress response by directly binding to promoters of stress resistant genes such as PdbHSP90-6.In addition,PdbCRF5 can interact with PdbCRF6,and PdbCRF6 also negatively regulates plant tolerance to salt stress,indicating that PdbCRF5 can also co regulate plant salt tolerance through interaction with other transcription factors.