Functional Analysis Of Bna.TTG2 Gene Family In Brassica Napus

Transcription factors (TFs) regulate gene expression by binding to specific cis-acting elements. Almost 70 transcription factor (TF) families have been identified in the well-sequenced model plant Arabidopsis, and they participate in almost all biological processes. The WRKY TFs are plant-specific and play roles in regulating several different plant processes, such as plant growth, development and stress response. However, few WRKY genes from Brassica napus (B. napus), an important amphidiploid oil crop, have been functionally studied. In this study, we identified the Bna. TTG2 gene family from B. napus, and further studied the gene function and regulatory mechanism of Bna.TTG2 genes in B. napus and Arabidopsis. The main results are as follow:1. According to the results of Southern blot and bioinformatics analysis, we identified four Bna.TTG2 loci in B. napus. Subsequently, we cloned four Bnα.TTG2 genes from the genome DNA and cDNA, respectively, of B. napus cultivar Jia 6104. Sequence analysis showed high levels of identity with the Arabidopsis TTG2 homologue.2. Four promoter fragments of Bnα. TTG2 family were cloned and fused to the GUS report constructs, and then transformed into wild-type (WT) Arabidopsis. GUS staining showed that all four Bna.TTG2 genes were highly expressed in roots, root hairs, trichomes, pollens, developing seeds and mature embryos. The results of subcellular localization indicated that four Bna.TTG2 proteins as well as the BnaA.TTG2.a.1△N protein (with the N-terminal 77 amino acids deleted) were localized in the nucleus. The transcriptional activation/repression assay using a Dual-luciferase reporter (DLR) system in Arabidopsis mesophyll cell indicated that a repression domain in the N-terminal region of residues 41-100 can suppress the activation function of the domain located at the residues 1-40.3. All four members of the Bnα.TTG2 family are capable of rescuing the trichome phenotypes of Arabidopsis ttg2 mutants. Overexpression of BnαA.TTG2.α.1 in Arabidopsis and B. napus remarkably increased the trichome number in transgenic plants, indicating that the Brassica TTG2 genes have a similar function as Arabidopsis TTG2 in trichome development. Complementary assay using series of splicing variants revealed that two WRKY domains are required for the full function of Bna.TTG2 genes.4. BnαA. TTG2.α.1-overexpressing Arabidopsis and B. napus plants showed increased sensitivity to salt stress, the growth of transgenic plant were significant reduced compared with WT under salt stress. However, we found that the osmotic stress and ionic toxicity do not contribute to the salt sensitivity of BnαA.TTG2.α.1-overexpressing plants. Furthermore, overexpression of BnaA.TTG2.a.1 might disturb starch metabolism under salt stress conditions.5. Our further study indicated that the endogenous IAA content was reduced and expression levels of two auxin biosynthesis genes as well as several auxin response genes were suppressed in BnαA.TTG2.α.1-overexpressing Arabidopsis under salt stress. Yeast one-hybrid and DLR assay uncovered that BnaA.TTG2.a.1 can bind directly to the promoters of two auxin biosynthesis genes, TRP5 and YUC2 in yeast and Arabidopsis mesophyll protoplasts. Arabidopsis plants overexpressing BnαA.TTG2.α.1△N were not hypersensitive to salt stress, suggesting a critical role of N-terminal region of BnaA.TTG2.a.1 in the salt stress response.6. We found that the survival rates of BnαA.TTG2.α.1-overexpressing Arabidopsis under glucose stress medium were significant higher than that of WT. In addition, overexpression of BnαA.TTG2.α.1 enhanced seed sensitivity to ABA in germination assays. This results indicated that Bnα.TTG2 genes may involve in glucose and ABA signal transduction pathway.7. Using a yeast two-hybrid screening, we identified 21 TFs that may interact with BnaA.TTG2.a.1. We further confirmed the interaction of BnaA.TTG2.a.1 and three putative proteins HDG1, HDG12 and BZIP43 by BIFC assays. We further found that BZIP43 may directly interact with the N-terminal region of BnaA.TTG2.a.1 protein and such an interaction could be influenced by salt stress in yeast cells.