Gene Regulatory Network Of Tamarix Hispida In Response To High Salt Or Drought Stress And Analysis Of Gene Salt Tolerance Function

Tamarix hispida is a strong tolerance to salinity and drought woody plant,and it is of great significance to study the resistance mechanism of T.hispida.Gene regulatory network(GRN)can clearly explain the relationships and roles of different genes in specific biological processes,which has guiding significance for identifying key regulatory genes to reveal the molecular mechanism of the biological processes.In this study,we established the GRNs of T.hispida in response to high salt or drought stress,and screened out the key regulatory genes that play a role in the process of both drought and salt tolerance.In addition,we obtained an ethylene insensitive 3-like(EIL)transcription factor which named ThEIL1 based on transcriptome data.In this study,the salt tolerance mechanism of ThEIL1 was studied using T.hispida as experimental material.T.hispida was treated with 500 m M Na Cl and 35% PEG6000 for 3,6,9,12 and 24 h,respectively,T.hispida was treated with water as a control.Different treatments were started at different times so that all treatments and control were collected at the same time,and 3biological replicates were performed for RNA-seq analysis.According to the transcriptome data,the gene regulatory networks(GRNs)of T.hispida in response to high salt or drought stress was established and identified by using the Pearson correlation coefficient、algorithm combining partial correlation coefficients and biological processes.The overexpression vector and knockdown vector of ThEIL1 were constructed and respectively transiently genetic transformed into T.hispida,and the overexpression vector of ThEIL1 was stably transformed into Arabidopsis thaliana using the floral dip method to obtain transgenic lines.Under salt stress,transiently genetic transformed T.hispida and transgenic A.thaliana were used as experimental materials to study the salt tolerance mechanism of ThEIL1.(1)Study on the GRNs of T.hispida in response to high salinity and drought stress.According to the transcriptome data,the differentially expressed genes(DEGs)in response to salt stress and drought stress were divided into regulatory genes and regulated genes.A bottom-up approach was used to establish a GRN,and the functional genes involved in biological processes response to salt and drought stress as the bottom layer.Transcription factors regulating these functional genes were predicted using Pearson correlation coefficient and algorithm combining partial correlation coefficients,and these TFs were served as the second layer of the GRN.Then,The second-layer of GRN TFs were used as research objects,and the Pearson correlation coefficient and partial correlation coefficient were used to predict the transcription factors that regulate these TFs and serve as the first layer of GRN.Thus,two three-layer GRNs of T.hispida in response to high salt and drought stress were obtained.(2)Validation of GRNs and analysis of gene stress resistance.The transcription factors in 26 GRNs were randomly selected and cloned,thus,5 transcription factors in the first layer and 6transcription factors in the second layer of GRN in response to Na Cl stress,5 transcription factors in the first layer and 10 transcription factors in the second layer in response to PEG stress.All TFs were constructed into p CM1307 vector containing Flag tag and transiently genetic transformed into T.hispida as study materials.ChIP-PCR and q RT-PCR were used to identify the regulatory relationship between transcription factors and predicted target genes.The results of ChIP-PCR and q RT-PCR showed that the regulatory relationship between transcription factors and their predicted downstream target genes was direct regulation,indirect regulation and non-regulation.According to the statistics of regulatory and non-regulatory relationships,more than 75.58% of the regulation was proved to be direct regulation and indirect regulation.The above results show that the GRNs are real and reliable.In addition,20 genes were randomly selected from these 26 transcription factors for transiently genetic transformed into T.hispida,and the physiological indexes related to stress resistance were determined to carry out stress resistance analysis.In order to further study the salt tolerance and drought resistance of transcription factors in GRNs,eight transcription factors were randomly and stably transformed into A.thaliana,and their salt tolerance and drought resistance functions were studied.The above results indicated that overexpression of these transcription factors significantly improved plant resistance.(3)Yeast one-hybrid system was used to obtain TFs that could regulate both the top-layer transcription factor of GRN under Na Cl stress and the top-layer transcription factor of GRN under PEG stress.A transcription factor was randomly selected from the top-layer of GRN under Na Cl stress and the top-layer of GRN under PEG stress,respectively,and the promoters of these two transcription factors were constructed into p HIS2 vector and co-transferred with the T.hispida c DNA library to Y187.After two yeast one-hybrid,the yeast plasmid was obtained and sent to the company for sequencing,and then a large number of TFs that can regulate the top-layer transcription factor of GRN under Na Cl stress and the top-layer transcription factor of GRN under PEG stress at the same time.Three transcription factors with higher enrichment number were selected from these TFs for ChIP-PCR verification.The results showed that these three transcription factors could regulate both the top transcription factors of GRN under Na Cl stress and the top transcription factors of GRN under PEG stress.(4)Study on the function and salt tolerance mechanism of ThEIL1 in T.hispida.Firstly,multiple sequence alignment and phylogenetic tree analysis of ThEIL1 protein were performed,and the results showed that ThEIL1 has a conserved domain of EIN3 and a highly conserved N-terminal amino acid sequence.The subcellular localization result showed that the protein was located in the nucleus.The yeast two-hybrid method was used to study whether ThEIL1 has transcriptional activation,and the result confirmed that ThEIL1 has transcriptional activation and the self-activating region is at the end of the gene.Under salt stress,the stress tolerance of the overexpression and knockdown of ThEIL1 transiently genetic transformed into T.hispida and the overexpression of ThEIL1 transgenic Arabidopsis were studied,and the results showed that overexpression of ThEIL1 significantly improved the salt tolerance of plants.The motifs bound by ThEIL1 were studied by using yeast one-hybrid and EMSA,and the results showed that ThEIL1 specifically recognizes PERE and GCC-box,but not bind to their mutants.The above results show that we have successfully constructed GRNs of T.hispida in response to high salt or drought stress,and identified the key genes regulating salt or drought tolerance,as well as the genes regulating salt and drought tolerance at the same time.And yeast one-hybrid system was used to obtain TFs that could regulate both the top-layer transcription factor of GRN under Na Cl stress and the top-layer transcription factor of GRN under PEG stress.ThEIL1 specifically recognizes PERE and GCC-box motifs to activate the expression of downstream target genes,thereby overexpression of ThEIL1 made plants decreased the damage of cell membrane and ROS content,and increased SOD and POD activities,proline content and trehalose content,thereby improving the salt tolerance of plants.In addition,when ThEIL1 was overexpressed and stably transformed into A.thaliana,the salt tolerance of transgenic A.thaliana was significantly improved.Furthermore,the salt tolerance mechanism of ThEIL1 was clarified.