Isolation And Characterization Of ZjDREB1 Gene Encoding A Dre-Binding Transcription Factor From Zoysiagrass (Zoysia Japonica)

Zoysiagrass (Zoysia japonica) is one of the major warm-season grasses widely utilized both at home and abroad, with excellent attributes of a very dense turf by means of slow growing underground rhizomes and rapid developing above-ground stolons, well adapted for lawns and golf turfs in the transitional and warm climatic regions, besides requiring minimal maintenance inputs. As the studies regarding zoysiagrass just got off to a late start in China, novel functional gene discovery and molecular breeding remain to be further researched. In order to reveal the molecular mechanisms involved in stress tolerance of turfgrass and obtain favorable genes of proprietary intellectual property, we isolated the ZjDREBl gene encoding a DRE-binding transcription factor from a cold-tolerant zoysiagrass cultivar on the basis of freeze tolerance evaluation, and analyzed its expression pattern as well as function. The main results are given as follows:1. Low temperature is one of the primary limiting factors for distribution and widespread use of released zoysiagrass(Zoysia spp.) cultivars in the transition zones and temperate regions. Three cultivars of zoysiagrass used in this study (Meyer, Palisades and Lanyin 3) were all introduced from the United States, and in common use both at home and abroad. Low-temperature tolerance was evaluated by measuring leaf electrolyte leakage (EL). The temperature at which 50% of the plants were killed (LT50) was determined. Our results showed that the lethal temperature (LT50) of the three zoysiagrass accessions predicted by EL was respectively -10.3℃in Meyer,-8.9℃in Palisades and -7.2℃in Lanyin 3. According to LT50 values, it was concluded that the freeze tolerance of the three cultivars ranked as:Meyer> Palisades> Lanyin 3.2. Dehydration-responsive element binding (DREB) proteins, specifically interacting with the DRE cis-acting element, have been identified as a group of important transcription factors that regulate the expression of many stress-inducible genes in plants. In this work, a novel DREB ortholog, designated as ZjDREBl (GenBank accession no.:GQ848096), from the cold-tolerant zoysiagrass cultivar Meyer was isolated using RT-PCR and RACE methods. It contained an open reading frame (ORF) of 774 bp encoding 257 amino acid residues. The predicted molecular mass of the deduced protein was 28.85 kDa and theoretical isoelectric point (pI) was 5.53. The deduced protein sequence featured a conserved AP2/EREBP DNA-binding domain, and comprised of a nuclear localization signal (NLS) and an acidic activation region, showing the typical characteristics of the DREB gene family. The phylogenetic tree constructed on the basis of amino acid sequences suggested that ZjDREBl from zoysiagrass and BeDREBl,2 from bermuda grass shared the highest 84% identity, and ZjDREB1 was classified into the A-2 group of DREB proteins. A comparison of the cDNA and its genomic counterpart (GenBank GU290546) demonstrated that the gene consisted of 2 exons and 1 intron. To our knowledge, this is the first report on isolation of DREB-like genes in zoysiagrass.3. In order to be used as an internal control in subsequent expression analysis of ZjDREB1, a 1560-bp full-length cDNA of actin was cloned from the leaves of zoysiagrass using RT-PCR and RACE methods. The sequence analysis revealed that it contained an ORF of 1134 bp encoding a protein composed of 377 amino acids, a 5’-UTR of 157 bp and a 3’-UTR of 495 bp. The predicted molecular mass of the deduced protein was 41.72 kDa and theoretical isoelectric point (pI) was 5.23. Homologous alignment showed that it shared over 85% of nucleotide identities and 97% of amino acid identities with actins from other plants in GenBank. The full-length cDNA was designated as ZjACT with its accession number GU290545. The phylogenetic tree constructed on the basis of amino acid sequences suggested that the relationship of ZjACT from zoysiagrass was most intimate with Hvactin from barley and TtACT-1 from poulard wheat, and they might have the same differential time in evolution. A comparison of the cDNA and its genomic counterpart (GenBank accession no. GU290546) demonstrated that the gene consisted of 4 exons and 3 introns. These results provided clues for studies on evolution of Actin gene family, and laid the foundation of researches into its functional and evolutionary diversity, as well as served as references for functional analysis and utilization of Actin gene from turf and forage grasses.4. Using Actin gene ZjACT as the internal control, expression profiling of ZjDREB1 genes from three zoysiagrass cultivars in response to cold was quantitatively analyzed by Real Time PCR. The results showed that there was no expression of ZjDREBl in room temperature. Its transcripts started to rise after 1 h at 4℃, rapidly and strongly up-regulated by chilling treatment, then reached a maximum level after 6 h of exposure to low temperature. The expression level of ZjDREB1 in the cold-tolerant cultivar was higher than that of the cold-susceptible one. A decreasing positive temperature resulted in higher accumulation of ZjDREBl transcripts with a dramatic increase below 8℃and peaking at 0℃. Further sequence analysis on ZjDREB1 genes from the three cultivars showed differences among cDNA sequence, genomic DNA sequence and the deduced amino acid sequence, inferring one of the possible reasons why their expression patterns differed significantly. Studies on the correlation of the function of the differentially expressed genes and the cold tolerance in different cultivars may provide some new insights into the molecular mechanism of zoysiagrass in response to cold stress.5. Based on the full cDNA sequence, two primers were designed with the flanking restriction sites of BamHⅠand HindⅢ. Using the zoysiagrass cDNA isolated previously as template, the complete coding sequence for the ZjDREB1 ORF was PCR amplified, and inserted into the prokaryotic expression vector pET-30a(+). Recombinant pET-ZjDREB1 vector was then introduced into Escherichia coli BL21(DE3) host strain to analyze its possible function under cold stress. A fusion protein about 36 kDa was expressed in E.coli cells harboring pET-ZjDREB1 after the induction of IPTG by SDS-PAGE analysis. Compared with control cells, those recombinant ones expressing ZjDREB1 fusion protein showed significantly improved cell viability at low temperature, implying that the protein may play an important role in resistance of E. coli (and probably other organisms) to low temperatures. It is concluded that ZjDREB1 is suggested to be potentially useful for improving plant tolerance to environmental stresses.6. The ZjDREB1 gene isolated from zoysiagrass was inserted into the plant expression vector pCAMBIA1301 to construct the recombinant pCAM-ZjDREB1 plasmid. It was introduced into Agrobacterium tumefaciens LBA4404 host strain, and then transformed into wild type Arabidopsis thaliana through floral-dip method. A total of 10 resistant plants were obtained in T0 generation via hygromycin screening. Among them 5 positive seedlings were further verified by PCR and semi-quantitative RT-PCR analysis. The results indicated that exogenous ZjDREB1 gene had been integrated into the Arabidopsis genome and expressed at the transcription level. Low-temperature tolerance was evaluated by measuring leaf electrolyte leakage. Our results demonstrated that transgenic plants overexpressing ZjDREB1 gene (LT50=-8.6℃) had a significantly greater cold tolerance than that of wild-type control plants (LT50=-5.5℃). It implied a promising future of the applications of ZjDREB1 gene in genetic engineering for stress tolerance improvement of turf and forage grasses.7. In order to improve stress tolerance of centipedegrass(Eremochloa ophiuroides), an important perennial warm-season grass, Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pCAMBIA1301 containing stress tolerance-related transcription factor ZjDREB1 gene from zoysiagrass was used to transform axillary bud-derived embryogenic calluses of the good selection ’E126’. So as to systematically optimize the conditions for centipedegrass transformation, several factors known to influence Agrobacterium-mediated DNA transfer were examined, including concentration of selective agent and bacteriostat, bacterial culture OD600, duration of infection and co-cultivation. Consequently, an efficient and reproducible transformation system for the production of transgenic centipedegrass plants was established. It was found that the efficiency of transformation highly relied on the following optimal conditions:30 mg/L hygromycin for selection,400 mg/L cefotaxime for bacteriostasis, bacterial culture OD600 of 0.1-0.3, infection for 30 min and co-cultivation for 3 d. Furthermore, the inclusion of 100 mM acetosyringone in both the infection and co-cultivation media led to an increase in transformation frequency. Thus, the hygromycin-selected plants were obtained and 5 putative transgenic plants were confirmed by PCR, semi-quantitative RT-PCR analysis and test of leaf-dip in hygromycin, preliminarily indicating the integration of exogenous ZjDREB1 gene into the genome of centipedegrass. The low-temperature tolerance assessment demonstrated that LT50 of transgenic plants overexpressing ZjDREB1 gene (-4.4℃) was significantly lower than that of wild-type control plants (-1.9℃). It suggested that the positive plants could be used for the subsequent studies on their stress tolerance and cultivation of new varieties of transgenic centipedegrass with improved resistance.8. For a better understanding of orther key genes from zoysiagrass in signal transduction pathways under stress conditions (e.g. COR, ICE, AREBIABF, MYC/MYB, bZIP), exploring molecular mechanism involved in stress response of zoysiagrass, and developing a functional genomics platform, the first cold- and drought-induced cDNA library of zoysiagrass was constructed. Plants of zoysiagrass were subjected to cold and drought stress respectively. Total RNA was extracted from their leaves. Then mRNA was isolated and purified, reverse transcribed into double-stranded cDNA. The directional cDNA library enriched for full-length sequences was constructed using Gateway(?) technology. The results indicated that the titer of the original library was 1.76×106 pfu/ml, the capacity was 7.04×106 pfu, and the average insert size was larger than 1 kb with the recombination efficiency of 90%. It is suggested that the cDNA library was successfully established in high quality, most likely containing lots of novel genes. Providing resources for zoysiagrass genomics, the library can be an effective tool for further studies on high-throughput EST sequencing, new stress-responsive genes screening, gene chips preparing etc.