Isolation And Function Analysis Of CN Gene And ERF Genes Related To Disease Resistance And Stress Tolerance In Plants

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.Tobacco is not only the model plant for genetic engineering but also an economic important crop.Tobacco mosaic virus(TMV) is the most widespread virus in tobacco,which is found in all countries where tobacco is grown.TMV severely reduces the yield and value of the cured tobacco.So it is significant to screen TMV-resistant tobacco cultivars,from which the resistant-related genes were isolate and expression pattern and function of the genes were analyzed.On the other hand,ERF transcription factors have been identified in different plant species,such as Arabidopsis,tomato,tobacco,and rice. 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.1),A resistance N gene homologous,designated CN gene,was cloned from the native Chinese tobacco germplasm HZNH plants by the technology of homology cloning,and analysis of expression pattern and function of the CN gene were carried out.2),The sequences containing the AP2/ERF domain were acquired from the TIGR soybean gene index database by Bioinformatics methods.Bioinformatics analysis of the AP2/ERF gene family and the analysis of expression pattern of the ERF subfamily unigenes were performed.The genes which had the better expression under the biotic and abiotic stresses were further analyzed.The detail contents of this research were as follows:1.Isolation and structure analysis of the CN gene:A resistance N gene homologous, designated CN gene,was cloned from the native Chinese tobacco germplasm HZNH plants by using the homologous cloning method.The coding region of CN gene is 3423 bp long and shared 93.63%nucleotide identity with the N gene.CN gene consists of 5 exons and 4 introns,the exon sequences are highly homologous to those of the N gene,however the introns significantly differ in length and sequence.Sequence analysis of the CN gene revealed that the CN gene belongs to TIR/NBS/LRR resistance gene class.2.Analysis of expression pattern and function of the CN gene:The expression of the CN gene was induced by TMV infection,which preceded the activation of the PR-1a genes and was temperature sensitive.No alternative transcription was produced from the intron 3 of the CN gene response to TMV-U1 infection. Organ-specific expression analysis suggested that CN transcripts accumulated at a high level in leaves,low level in stems and minim level in roots.Transforming CN gene into TMV-susceptible N.tabacum cv.K326 plants by Agrobacterium-mediated method,transgenic K326 plants displayed HR and systemically hypersensitive response(SHR).3.Bioinformatics analysis of the AP2/ERF gene family in soybean:148 TC/EST sequences containing the AP2/ERF domain were acquired from the TIGR soybean gene index database.Thereinto,26 unigenes were assigned to the AP2 family,2 unigenes were assigned to the RAV family and 120 unigenes were assigned to ERF family.98 of these 120 unigenes in the soybean ERF family were predicted to encode a complete AP2/ERF domain and were further analyzed.An investigation of phylogenetic relationship and the sequence conserved motifs in soybean ERF family proteins was performed and compared with those of Arabidopsis.As a result of these analysis,the ERF family in soybean was divided into 12 subgroups,A1-A6 and B1-B6,and the classification used for Arabidopsis was also applicable to the soybean ERF family.The AP2/ERF domain was much conserved in the soybean and Arabidopsis,outside the AP2/ERF domains,there had many soybean-specific conserved motifs which may play important roles in regulating certain biological processes in soybean,besides some common conserved sequence motifs.According to described Arabidopsis ERF classification of Sakuma et al.(2002),the members in the A1-A6 subgroups belong to DREB subfamily and the members in the B1-B6 subgroups belong to ERF subfamily.4.The expression pattern of the ERF subfamily unigenes:the expression patterns of 10 unigenes belonging to the 6 different subgroups of the ERF subfamily were analyzed under various treatments.Inoculation with SMV as biotic stress increased the transcript levels of 7 tmigenes in soybean plants.Drought,low temperature and high salinity as abiotic stresses,induced the expression of 10,6,and 10 unigenes, respectively.The expression of all the 10 tested unigenes was induced by treatments with SA,ET,JA and ABA.5.Analysis of DNA-binding specificity of the GmERF3 and GmERF4 genes in vitro: 2 ungenes of the above 10 tested ungenes who had the better expression under the biotic and abiotic stresses,designated GmERF3 and GmERF4,respectively,and were used for further analysis.The results of electrophoretic mobility shift assay (EMSA) indicated that GmERF3 and GmERF4 specifically bound to DRE (dehydration- responsive element) and ERE(ethylene-responsive element or GCC-box element) in vitro.Furthermore,when a mutated version of the GCC box (mGCC) and DRE(mDRE) were used in the assay,the mobility shift was not observed.The results suggested the two ERF genes may be involved not only in biotic stresses,but also in abiotic stress-induced signaling pathway.6.Subcellular localization assay of the GmERF3 and GmERF4 genes:In order to detect the subcellular localization of ERF transcription factor proteins,GmERF3 and GmERF4 were fused to the 5' terminus of the coding region of green fluorescence protein(GFP) under the control of the cauliflower mosaic virus (CaMV) 35S promoter.Then the fused plasmids were introduced into onion epidermal cells using a particle bombardment method with a PDS1000/He. Transformed cells were incubated for 24 h at 22℃in the dark and localization of the fusion proteins was then detected using a confocal microscope equipped with appropriate filter.The subcellular localization assay indicated that GmERF3 and GmERF4 can localize into the nuclei.7.Analysis of the autonomous activation of the GmERF3 gene:The modification site of GmERF3 protein was predicted by website database.According to a result of the prediction,the GmERF3 gene was divided into four fragments,and then these fragments were inserted into bait-vector(pGBKT7)of the yeast two-hybrid system for identifying antonomous activation.The results suggested yeast cells carrying the four fragments of the GmERF3 gene could grow on SD/-Ade/-His/-Leu/-Trp medium,respectively.Further analysis suggested those yeast cells could also grow on SD/-Ade/-His/-Leu/-Trp medium with 5mM 3-amino-1,2,4-triazole(3-AT), even 15mM 3-AT.8.Function analysis of the GmERF3 and GmERF4 genes:GmERF3 and GmERF4 were respectively transformed into tobacco plants(W38) by Agrobacterium-mediated method.The results suggested overexpression of GmERF3 enhances salt and drought tolerance in transgenic tobacco plants.On the contrary, the 35S::GmERF3 tobacco plants did not exhibit any detectable tolerance against cold stress.Furthermore,overexpression of the GmERF3 gene enhanced resistance to bacterial pathogen Ralstonia dolaanacearum and the fungal pathogen Alternaria alternata(Fries) Kissler in transgenic tobacco plants.When wild-type and GmERF3 transgenic tobacco plants were inoculated with TMV,all transgenic lines tested had no exhibited significantly reduced lesion size and leaf lesion numbers compared to wide type plants in the local infected leaves,but the collapse time of the apex of infected transgenic plants were obviously delayed than that of the infected wild-type plants.Overexpression of GmERF4 genes also enhances salt and drought tolerance in transgenic tobacco plants,but not exhibit any detectable tolerance against pathogen infection.Sum up,the CN gene and 2 ERF transcription factor genes were isolated and their molecular characterizations were analyzed.Transgenic tobacco overexpression the CN gene and 2 ERF transcription factor genes improved resistance to biotic or abiotic stresses.The isolation of the CN gene and ERF genes provided candidate genes for improving crop tolerance.