Molecular Cloning And Expression Analysis Of GmJERF3s Transcription Factor And GmIMTs Salt Stress Response Related Genes From Soybean

Plant growth and productivity are strongly influenced by biotic stress such as Pathogen infection and abiotic stress such as drought, high salt content and Temperature change. It is of important significance to isolate stress tolerance-related genes and gain stress-tolerant crops by transgenic technologies. The ERF transcription factors have been identified in different Plant species, such as Arabidopsis, tobacco, rice, and tomato. They Participate in the regulation of gene expression to drought, high salt content, low temperature, disease and cell developmental processes. The AP2/ERF transcription factor genes as regulative gene can induce expression of a number of downstream stress-related genes, resulting in enhanced resistance against biotic and abiotic stresses. It has extensive application value to isolate stress tolerance-related genes of transcription factors and gain stress-tolerant crops by transgenic technologies.In this research, the plant stress tolerance-related genes were investigated in two ways.l), three resistance transcription factor, was cloned from the Glycine max by the technology of homology cloning, and analysis of expression pattern and function of the GmJERF3s genes were carried out.2), In the same way as the cloned two by salt, drought stress inducible expression of Imtl genes. Specific content is as follows:1. In this study we characterized genes by the technology of homology cloning. Nucleotide sequence of JERF3was used as a querying probe to screen EST database o; Glycine max and the candidate ESTs was contigging.the extension of genes from genomic library of Glycine max. We made the gene-specific primers, three full-length cDNAs sequences of Glycine max were confirmed by amplifying cDNA of Glycine max. The full length of GmJERF3a was1.356bp. It contained a1,038bp open reading frame, encoding a polypeptide of345amino acids; The full length of GmJERF3b was1,322bp. It contained a1,149bp open reading frame, encoding a polypeptide of345amino acids; The full length of GmJERF3c was1,556bp. It contained a1,179bp open reading frame, encoding a polypeptide of392amino acids. The phylogenetic relationship also appeared that GmJERF3s protein was more similar to MtERF protein than other. And amino acid sequence alignment revealed that the homology was99%between GmJERF3s and MtERF. To investigate the expression pattern of GmJERF3s in Glycine max, the expression of the three genes at different organs was analyzed, in root、leaves of25DAF these genes were highly expression, but with no expression in other organs almost by real-time fluorescence quantitative PCR technique.2. The full length of GmIMT1was1,208bp. It contained a1,098bp open reading frame, encoding a polypeptide of365amino acids; The full length of GmIMT2was1,138bp. It contained a1,098bp open reading frame, encoding a polypeptide of365amino acids; The phylogenetic relationship also appeared that GmIMTs protein was more similar to Aaim tand Llimt protein than other. And amino acid sequence alignment revealed that the homology was99%between GmIMTs and Aaimt or Llimt. To investigate the expression pattern of GmIMTs in Glycine max, the expression of the two genes at different organs was analyzed. In root、leaves and stem these genes were highly expression, but with no expression in seed almost by real-time fluorescence quantitative PCR technique.3. To further examine the effect of GmJERF3s and GmIMTs genes, were cloned into the pEarlygate expression plasmid vector with correct orientation and size respectively, we obtained the plant expression plasmid pEarlygate-GmJERF3a、pEarlygate-GmJERF3b、 pEarlygate-GmJERF3c、pEarlygate-GmIMTl and pEarlygate-GmIMT2successfully.The pEarlygate-GmJERF3s and pEarlygate-GmIMTs construct was transformed to Arabidopsis thaliana by agrobacterium-mediated transformation system. After the preliminary selection of Bar, the pcr results of the anti-kan regenerated plantlets showed that the GmJERF3s and GmIMTs genes were transferred into Arabidopsis thaliana.4. Functional analysis of GmJERF3s and GmIMTs genes transgenic Arabidopsis for stress tolerance. Germination rates of GmJERF3s and GmIMTs genes transgenic Arabidopsis was obviously higher than that of wild-type Arabidopsis at high salinity ABA and mannitol stress. These results showed that over-expression of GmJERF3s and GmIMTs genes inereased salt、ABA and drought stress tolerance of Arabidopsis.