| Transcription factors can activate or repress the transcription of downstream genes by specificly combining with cis-acting elements in the promoter of target genes in the form of trans-acting factors, regulating the expression of related genes by interaction with other proteins. ERF transcription factors are plant-specific family genes related to plant growth and abiotic/biotic stress responses. RNA-Seq analysis of P. simonii×P. nigra leaves was conducted for the screening of poplar salt-induced ERFs, the results showed that the relative expression level of ERF76 gene is the most significant among all the up-regulated ERF family genes.The temporal-spatial expression pattern of ERF76 gene in three tissues across five time points by RT-qPCR displayed that ERF76 gene is induced by salt stress in root, stem, and leaf tissues, and the relative expression level is highest in root, followed by leaves and stem. During 3-36h period, the relative expression level of ERF76 gene reaches the plateau at 12h in all the tissues.The 1.5 kb upstream DNA sequence of ERF76 gene was cloned from P. simonii X P. nigra by nested PCR. The cis-element prediction showed that ERF76 promoter sequence contains transcriptional regulating elements, stress responsive cis-elements and plant growth and development associated cis-elements. The ERF76 promoter sequence was identified to be interacted with the proteins involved in transcription, plant defense and stress response, oxidoreductase activity, plant growth and development by yeast one-hybrid assay. And the expression levels of GUS driven by different promoter segments in transgenic Arabidopsis proved that the promoter sequences in different length possess different transcriptional activity and responded to salt stress at different degree by RT-qPCR, GUS-histochemistry staining and quantitative fluorometric GUS assay.The 1537 bp cDNA fragment encoding ERF76 gene was cloned from P. simonii×P. nigra by RT-PCR. Subcellular localization results indicated that ERF76 protein is targeted to the nucleus. The transgenic tobacco and poplar lines overexpressing ERF76 gene driven by cauliflower mosaic virus 35S promoter were obtained by Agrobacterium mediated leaf disc transformation method. Under salt stress condition, transgenic tobacco and poplar plants show significant increases in plant height, root length, fresh weight, antioxidase activity.stress responsive protection molecules content and relative water content, compared to wild type plants. And the accumulation of plant reactive oxygen species and relative electrical conductivity in transgenic lines are lower than those in wild type plants. There are significant phenotypic differences between transgenic poplar and wild type plants including leaf shape and root lenghth, and the relative contents of ABA and GA in transgenic poplar are significantly higher than those in wild type. The mRNA abundances of ERF76 gene and a few genes involved in stress responses and ABA/GA biosynthesis in transgenic poplar are significantly higher than those in non-transgenic plant. RNA-Seq analysis between transgenic poplar and non-transgenic plant showed that there are 375 differentially expressed genes (DEGs) containing 268 up-regulted genes and 107 down-regulated genes in transgenic poplar, compared to wild type. And the up-regulted genes incudes 16 transcription factor genes and 45 stress-related genes.The above indicated that poplar transcription factor ERF76 gene is highly up-regulated by salt stress, and plays an important role in plant growth and development, and salt stress responses. The overexpression of ERF76 gene can promote the growth and development and improve the salt tolerance of transgenic plants by regulating the expression of genes involved in plant hormone biosynthesis and stress-related genes in transgenic plants. |
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