Study On The Difference Of Potato Cadmium Accumulation Regulated By Potato Transcription Factor StWRKY6 And StHIPP1 Protei

Guizhou is one of the largest karst areas in the world and belongs to a high geological background of the heavy metal Cadmium(Cd),which is also one of the main potato(Solanum tuberosum L.)growing areas.WRKY transcription factors are one of the largest families of transcriptional regulators in plant research.The group selected "Yunshu 505",a variety with low Cd accumulation,from 100 potato varieties.Analysis of genome-wide data from potato revealed that the key gene responsible for the differences in Cd accumulation and tolerance among cultivars was the transcription factor StWRKY6.Heterologous expression of StWRKY6 into Arabidopsis thaliana was found to have enhanced Cd tolerance in the plants.Therefore,this work was carried out to analyze the transcriptome under Cd stress from StWRKY6 heterologous expression into Arabidopsis,to explore the potential regulatory network of StWRKY6,and to perform screening,identification and functional validation of StWRKY6 proteins interacting with each other under Cd stress.A series of candidate proteins were first screened for possible interactions with StWRKY6 using gene co-expression network,interaction network prediction and Pull down mass spectrometry,and then the candidate proteins were further screened based on annotation information,literature reports and bioinformatics.Finally,yeast twohybrid cotransformation assay and bimolecular fluorescence complementation assay were used to verify its interactions with StWRKY6,followed by functional complementation assays to test the Cd tolerance and accumulation ability of the Cdsensitive Δycf1 strain by transforming the interacting protein,in order to provide some theoretical basis for revealing the molecular mechanism of StWRKY6 involved in the Cd tolerance mechanism of potato,and the results obtained are as follows.(1)Transcriptome analysis of genes associated with heavy metal Cd was performed on four leaf samples(WT-CK,WT-Cd,OE-CK and OE-Cd)of Arabidopsis thaliana after heterologous expression of StWRKY6.A total of 304 significantly differentially expressed genes(DEGs)were screened,including 74 differentially expressed genes in the CK group and up to 230 differentially expressed genes after 100 μM Cd stress treatment;DEGs were significantly enriched to photosynthesis,sulfur metabolism,phosphorylation pathway,etc.;some genes related to heavy metal Cd were identified related genes,such as WRKY,HIPP,ARR,TIFY gene family,etc.Screening of StWRKY6 candidate interactive proteins and their bioinformatics analysis.The STRING website prediction and Pull down-MS technology were used to screen 10 candidate interaction proteins StHBP2,StGTPR2,StATPF4,StMnSOD,StTIFY6,StARR5,StWRKY40,StWRKY41,StPGK1 and StFBA1.It was predicted that StWRKY6 may interact with heme protein HBP family,jasmonic acid-ZIM zinc finger TIFY family,superoxide dismutase MnSOD family,etc.(2)Validation of StWRKY6 candidate interaction proteins.Ten pairs of proteins that could interact with each other were verified by Y2 H and BiFC,and finally it was determined that the potato transcription factor StWRKY6 interacted with four proteins,StMnSOD,StHBP2,StGTPR2 and StHIPP1.(3)Validation of StWRKY6 candidate reciprocal proteins.Ten pairs of potentially interacting proteins were verified by Y2 H,BiFC,and finally the potato transcription factor StWRKY6 was identified to interact with four proteins,StMnSOD,StHBP2,StGTPR2 and StHIPP1.(4)Functional validation of StWRKY6-interacting proteins StMnSOD,StHBP2 and StHIPP1.Real-time fluorescence quantification results showed that StMnSOD and StHBP2 reached the highest expression at a concentration of 25 μM after 12 h of different Cd stresses,indicating that StMnSOD and StHBP2 responded to Cd.Functional complementation validation of yeast mutants showed no significant difference in the growth of yeast cells without Cd treatment.StHIPP1 improved the sensitivity of Δycf1 to Cd when 50 μM Cd was provided to the medium.It was tentatively demonstrated that StHIPP1 has a positive regulatory effect on yeast Cd stress.In this study,StWRKY6 protein and StHIPP1 protein interact to improve the cadmium tolerance of potato.The results of this study enrich the research of potato WRKY family proteins,lay a foundation for elucidating the interaction mechanism of potato under Cd stress,and provide clues for the role of StWRKY6 in Cd resistance defense response and signal transduction pathway.