Functional Analysis Of Maize Stress Response Genes ZmUBP And Evolutionary Analysis Of WRKY Gene Family

Abiotic stress is a restricting factor for the growth of crops, and often causes very serious reduction of the crop yields. In recent years, maize (Zea mays L.) has become one of the world’s most widely cultivated gramineous plant increasingly. To improve the quality of the crops, resistance breeding has been developed as an important direction. The researches on the functions of stress related proteins and the application of these molecules in the breeding of new varieties which can adapt the abiotic stresses have becoming efficient ways to increase maize outputs. Ubiquitin-Specific Proteases (UBP) are highly conserved proteins on phylogenesis and exist extensively in eukaryotes, and their main functions are focused on the regulation of cell activity and plant growth by de-ubiquitination. They also play some important roles on the tolerance of plants to environmental stresses. But so far, the function of these proteins in maize has not been clarified. In this study, ZmUBP14 (accession number:GRMZM2G107696) was identified as the homolog of AtUBP 14 in maize by amino acid BLAST, and ZmUBP16a (accession number:GRMZM2G136443), ZmUBP16b (accession number: GRMZM2G388987) and ZmUBP16c (accession number:GRMZM2G116314) were identified as the homolog of AtUBP16. The structure features and expression patterns of these four molecules were performed to illustrate their functions in the resistance to abiotic stresses. We also analysed the molecular evolution of WRKY transcription factor family of maize and other gramineae species. And the main results are as follows:1, Protein structure analysis of ZmUBP 14 and ZmUBP 16a/b/c showed that they all contained the UBP domain. Tissue-specific expression indicated ZmUBP 14 and ZmUBP 16a/b/c were highly expressed among all tissues and stages, which implied they had crucial functions over the whole life cycle of the maize probably. Further expression analysis showed that the expression level ZmUBP16a, ZmUBP 16b and ZmUBP 16c could be induced by abscisic acid (ABA), and reduced by CdCl2 and NaCl. ZmUBP 14 and ZmUBP 16a/b/c were cloned and sequenced, and the results shared 100% sequence identity with the predicted sequence in the maize B73 annotated database. Subsequently, they were cloned and fused to GFP-tagged plasmid to investigate the protein subcellular localization. Overexpression of ZmUBP-GFP fusion proteins in Arabidopsis indicated that ZmUBP14 protein was localized in the cell nucleus, while ZmUBP16 a/b/c-GFP fusion proteins were localized in the plasma membrane.2、For the study of the protein function of ZmUBP 14, Zm UBP14 overexpression vector was constructed and transformed into Arabidopsis ubpl4 mutant plants. We found that the leaf morphology of Arabidopsis ubpl4 mutant could be recovered by ZmUBP 14 transformation. Pull-down assay suggested that ZmUBP 14 could interact with transcription factors like ZmTCP15a and ZmTCP15b in vitro. A bimolecular fluorescent complementation (BiFC) assay performed in tobacco mesophyll cells confirmed the interactions between ZmUBP 14 and the two transcription factors.3、Zm UBP16a/b/c overexpression vectors were constructed and transformed into Arabidopsis ubpl6 mutant plants, and the salt intolerance could be recovered by the overexpression of ZmUBP16a, ZmUBP16b or ZmUBP16c in ubpl6 Arabidopsis mutant. The overexpression of ZmUBP16a/b/c in wild-type Arabidopsis didn’t enhance the tolerance to salt stress, but improve the ABA sensitivity of transgenic Arabidopsis. Meanwhile, the heavy metal tolerance of transgenic plants could be increased by the ZmUBP16a/b/c overexpression.4、The expression levels of some stress-responsive genes in ZmUB 16a/b/c overexpression lines were up-regulated significantly compared with the wild-type ones. The relative transcription levels of the genes related to Cd tolerance, such as AtABCCl, AtABCC2, and AtPCS1 were up-regulation after CdCl2 treatment, and the induced levels of these genes were much more remarkable in the ZmUBP 16a/b/c overexpression plants. The expression levels of ABA signaling pathway related genes like AtNCED3, AtMYB2 and AtRD22 were also induced in the transgenic plants, while ABA independent gene AtCBF didn’t change significantly after ABA treatment.5、In the study, we adopted a high-throughput RNA-seq approach to determine the function of ZmUBP 16a under heavy metal stress. Four transcriptome libraries were constructed from Arabidopsis wide-type and ZmUBP 16a overexpression transgenic plants that had been treated with or without 10μM CdCl2, respectively. Gene differential expression analysis revealed that transcripts related to metabolism, including Energy metabolism, lipid metabolism, carbohydrate metabolism, amino acid and nucleotide metabolism were activated by the overexpression of ZmUBP16a. After the heavy metal treatment, these metabolic-related genes were suppressed in both wide-type and ZmUBP 16a overexpression plants. Three genes were up-regulated by both the gene overexpression and heavy metal treatment. One of them is AtUGT74E2, which was reported as an important gene in reactive oxygen species (ROS) and plant hormone signal transduction pathways.6^ In gramineous species, tandem duplication events played the leading role in gene expansion of WRKY transcription factors family. Highly conserved regions of microsynteny were also found in the four Gramineae species. All 37 segments formed 8 groups of orthologous segments. In addition, the selection pressure analysis showed that purifying selection affected the evolution of WRKY family in gramineae.