Isolation And Characteirzation Of ERF Transcirption Factors, GmERF6and GmERF7, From Soybean (Glycine Max L.)

Soybean is one of most important grain and commercial crop. It is not only themain source of protein and lipid for human, but also the important cattle fodder andindustrial raw material. However, the adverse environmental factors, such as disease,drought, cold and soil salinization, seriously affect the growth and development ofsoybean which bring terrible loss to it. It was reported that ERF transcription factorsplayed vital roles in response to biotic and abiotic stresses, downstream geneexpression regulation and improvement of anti-adversity in plant. In this paper, twonew ERF transcription factors, GmERF6and GmERF7, were cloned from soybeanJilin32. The function and regulation mechanism of them were analyzed in order toprovide theoretical bases for application and enrich the gene resource of ERF. Theexperiment results are following:1. GmERF6and GmERF7were cloned by RT-PCR in soybean Jilin32cDNA. TheORF of GmERF6is582bp encoding a polypeptide of260amino acids. The predictedGmERF6protein contained a conserved AP2/ERF domain, two putative nuclearlocalization signal sequences and a repression-associated EAR motif which suggestedthat GmERF6may be acted as a transcription represser in the nucleus. The ORF ofGmERF7is1179bp encoding a polypeptide of392amino acids. The predictedGmERF7protein contained a conserved AP2/ERF domain, two putative nuclearlocalization signal sequences, an putative activation domain and a conservedN-terminal MCGGAI(I/L) motif which suggested that GmERF7may be acted as atranscription activator in the nucleus. The amino acid sequence alignment andphylogenetic tree analysis indicated that GmERF6was grouped into Class II in ERFtranscription factors subfamily and was related most closely to the GmERF4insoybean, implying that they may be diversified from a common ancestor during evolution; GmERF7was grouped into Class IV in ERF transcription factorssubfamily and was related most closely to the GmERF7in soybean, implying thatthey may be diversified from a common ancestor during evolution.2. The green fluorescent protein fused expression vectors of GmERF6and GmERF7were constructed and transformed into onion epidermal cells usingAgrobacterium-mediated transient expression. The results indicated GmERF6andGmERF7were both localized in the nucleus.3. GmERF6and GmERF7were expressed in yeast to analyze their transcriptionalactivation. The results indicated GmERF6had no transcriptional activation ability inyeast; however, GmERF7had transcriptional activation ability in yeast whichsuggested it is a transcription activator.4. GmERF6and GmERF7were expressed in procaryotic cell and the recombinantproteins of them were purified to perform electrophoretic mobility shift assays. Theresults indicated GmERF6and GmERF7were both able to specifically bind to theGCC-box.5. The regulation abilities of GmERF6and GmERF7were analyzed in tobacco leavesby transient expression. The results indicated GmERF6had transcription repressedability which repressed not only the expression of downstream genes but also theactivity of other transcriptional activators; GmERF7had transcription activated abilitywhich activated the expression of downstream genes.6. The expression patterns of GmERF6and GmERF7were analyzed usingquantitative real-time PCR. The results indicated GmERF6and GmERF7had notissue-specific expression characteristics which expressed in roots, stems, leaves,flowers and immature embryos. Their expressions were both higher in roots than inother tissues. GmERF6and GmERF7responded differently to biotic and abioticstresses such as drought, salt, cold, wounding, ABA, ET, SA and MeJA.7. The promoter sequences of GmERF6(1986bp) and GmERF7(1963bp) wereisolated from the soybean genome using PCR. Sequence analysis indicated there wereseveral types of putative stress-related cis-elements in GmERF6P and GmERF7P. TheGmERF6P and GmERF7P were cloned into pCAMBIA1301and than transformed into tobacco leaves using Agrobacterium-mediated transient expression. The resultsindicated that GmERF6P and GmERF7P both had weak ability to promote genes toexpress. The promoter activity of GmERF6P was activated differently by drought, salt,cold, ET, GA and ABA treatments for10h; The promoter activity of GmERF7P wasactivated differently by drought, salt, ET and GA treatments for10h, however, it wasrepressed by cold and ABA treatments for10h. The promoter activity of GmERF6Pwas repressed when GmERF6P and GmERF6were cotransformed into tobacco leaveswhich indicated that there existed feedback inhibition in the expression of GmERF6.8. The coding region of GmERF6was cloned into pBI121under the control of theCaMV35S promoter. The pBI121-GmERF6plasmid was introduced into Arabidopsisusing Agrobacterium-mediated transformation following the floral dip method. Theexpression of stress-relate genes in T3GmERF6transgenic Arabidopsis were detectedby semiquantitative RT-PCR. The results indicated that the expression of AtKin1,AtSOS1, AtPR3, AtRD22, AtERF4, AtERF7and AtNIMIN1decreased significantly,however, the expression of AtPDF1.2and AtPR4increased. The seed germinationrates of GmERF6transgenic Arabidopsis were significantly higher than that of wildtype Arabidopsis. GmERF6transgenic Arabidopsis grew much healthier thanwild-type Arabidopsis removing water for18d. The water loss rates of detachedleaves of GmERF6transgenic Arabidopsis were lower than that of wild typeArabidopsis. These data indicated that transgenic Arabidopsis plants constitutivelyexpressing GmERF6showed an increased tolerance to drought compared to wild-typeplants.9. The coding region of GmERF7was cloned into pBI121under the control of theCaMV35S promoter. The pBI121-GmERF7plasmid was introduced into Arabidopsisand tobacco, respectively. The expression of stress-relate genes in T3GmERF7transgenic Arabidopsis were detected by semiquantitative RT-PCR. The resultsindicated that the expression of AtKin1and AtRD29A significantly increased. Theseed germination rates of GmERF7transgenic Arabidopsis were significantly higherthan that of wild type Arabidopsis. GmERF7transgenic tobacco plants grew muchhealthier than wild-type tobacco under salt treatment. GmERF7transgenic tobacco plants maintained higher levels of chlorophyll and soluble carbohydrates and lowerlevel of malondialdehyde compared with the levels in wild-type tobacco plants. Thesedata indicated that transgenic Arabidopsis and tobacco plants constitutivelyexpressing GmERF7showed an increased tolerance to salt compared to wild-typeplants.