Homologous Sequence Cloning Of Ta14S Gene In A,B,D Genomes And Their Expression Characteristics In Wheat

Drought,high temperature and low temperature are important abiotic stress factors limiting the yield and quality of wheat.Therefore,cloning key genes related to drought,heat and other abiotic stresses and analysing their expression characteristics are of great significance to further understanding molecular mechanism of stress tolerance and improving varieties in wheat.14-3-3 proteins are a family of ubiquitous eukaryotic regulatory molecules and involved in a series of signal transduction and regulation,may play an important role in response to environmental stresses.In order to investigate the regulation role of 14-3-3 protein in response to adversity stress in wheat,in this study,three homologous sequences of Ta14S gene were identified in wheat,and the structures,chromosome localization,expression pattern and subcellular localization of its coding protein and function were studied.The main results were as follows:1.Separation and chromosomal location of the three homologous sequences of Ta14S geneThe cDNA sequences of three Ta14S homologous genes were obtained by RT-PCR and RACE technique.The sequence analysis showed that the full-length c DNAs of the three homoeologous genes are 1294 bp,1267bp and 1189 bp,including the 196,160 and 82 bp of 5?UTR,306,315 and 315 bp of 3?UTR,respectively.The ORF of the three homoeologous genes are all 792 bp in length,26 nucleotide differences are found in the coding region.Compared to Ta14S-1,Ta14S-2 contained a 45 bp deletion and a 9 bp insertion in the 5 'UTR,Ta14S-3 contained a 22 bp and a 92 bp deletion in the 5' UTR.In the 3'UTR area,Ta14S-1,Ta14S-2 and Ta14S-3 contained 3,4 and 5 TGG repeat,respectively.Meanwhile,the genome sequences of three homologous genes of Ta14S were also seperated by PCR technology.Sequence comparison between c DNA and genome sequences isolated that Ta14S-1,Ta14S-2 and Ta14S-3 have very similar gene structure and all containing 5 exons and 4 introns.The third intron of the three Ta14S genes are 1148 bp,1148bp and 926 bp in length,respectively,and other introns are relatively conservative.According to the 3 'UTR seguence of three Ta14S homologous genes,the specific primers were designed and using the nulli-tetrasomic lines of Chinese Spring as the template.Three Ta14S homologous genes are located in wheat the second chromosome by PCR analysis.Thus the three Ta14S homologous genes were named Ta14S-2A,Ta14S-2B and Ta14S-2D,respectively.2.Amino acid sequence analysis of three Ta14S homologous genesThe amino acid sequences alignment showed that the amino acid sequences of Ta14S-2A,Ta14S-2B and Ta14S-2D were basically identical except site 241 and site 257.In the two sites,Ta14S-2A,Ta14S-2B and Ta14S-2D are glycine(gly,G)and asparagine(Asn,N),aspartic acid(asp,D)and histidine(His,H),aspartate(asp D)and asparagine(ASN),respectively.The phylogenetic tree analysis suggested that Ta14S-2A,Ta14S-2B and Ta14S-2D proteins have the closest genetic relatives with barley Hv14 A protein and they are all belong to non-epsilion group.3.Expression patterns of the three homologous genes of Ta14SThe expression patterns of each homologous genes in different growth stages of wheat organization and under PEG-stress were analyzed by real-time PCR.The results showed that transcription could be detected in all tissues.the expression level of Ta14S-2A,Ta14S-2B and Ta14S-2D are the highest in primary roots 1day after germination and the transcript level of Ta14S-2B was higher than that of Ta14S-2A,2D.The lowest expression was obsearved in embryos of mature seeds.Under PEG-treatment,the expression pattern of the three homologous genes were different in leaf and root.The expression levels of three homologous genes were higher in leaves than that of in roots.Moreover,the expression of three homologous genes were all significantly upregulated within 12 h under PEG treatment in the drought-resistant variety ‘Luohan No.2',but slightly down-regulated in Non-drought-resistant variety ‘Chinese Spring'.4.Subcellular localization of Ta14S proteinIn order to investigate the subcellular localization of Ta14S-2B protein,Ta14S-2B:GFP fusion expression vector was constructed and introduced into Arabidopsis protoplast by transformation with PEG-mediated.The transformation Arabidopsis protoplast cell were obsearved under the confocal laser scanning microscopy.The results showed that the fluorescence signal was mainly distributed in the cell membrane and cytoplasm and had no overlap with the chloroplast autofluorescence,speculating Ta14S protein was located in the cell membrane and cytoplasm but not in chloroplast.5.The construction of Ta14S-2B overexpression and RNAi expression vectors and obtains of the transgenic rice plantsTo study the role of Ta14S in wheat adversity stress response,overexpression and RNAi vectors of Ta14S-2B were constructed and introduced into rice callus by Agrobacterium-mediated transformation with japonica rice variety ‘Kitaake' as the receptor and 13 overexpression and 13 RNAi transgenic lines were obtained,respectively.PCR analysis showed that 24 positive plants were obtainted by detecting T1 generation seedlings,among which 12 overexpression and 12 RNAi.6.ABA sensitivity test of Ta14S RNAi transgenic rice linesT0 generation of Ta14S RNAi transgenic rice seeds were put into MS culture medium with 0,l,2,3?M ABA and seed germination rate was counted every day after seed germination.The results showed that the germination rate of transgenic rice seeds were significantly lower than controls,suggesting RNAi transgenic rice seeds were more sensitive to ABA during germination.When rice seedlings were grew in hydroponic nutrient solution with 3?M ABA treatment,the growing situation of RNAi transgenic seedlings with Ta14S were significantly weaker than control,indicating the seedlings also was more sensitive to ABA.