Analysis Of CsCBF2 Transgenic Tobacco Under Abiotic Stress

In the agricultural production,plants are often affected by various adverse environmental factors,among which low temperature is a serious environmental stress factor impacting on crop growth.CBF gene plays a key role in response to low temperature stress in plant.A CBF gene,CsCBF2,has been cloned from the tea tree in our laboratory,and its expression and function in regulating gene expression have been studied.However,its role in tolerance of abiotic stresses has not been elucidated.In this study,three transgenic tobacco lines with high ectopic overexpression of CsCBF2 were selected,and their tolerance to abiotic stresses was studied.Results from these experiments were summarized as follows:1.The T2 homozygous transgenic plants stably expressing CsCBF2 were screened by PCR,RT-PCR and Western blot analysis for the study of the role of CsCBF2 in the tolerance to low temperature,high salt and dehydration stress.2.The CsCBF2 transgenic plants were treated by salt,dehydration,and low temperature stress,and the phenotype was studied by determining germination rate,root length,fresh weight,survival rate and electrolyte leakage rate.Under the condition of high salt and dehydration stress,the germination time of transgenic tobaccos was earlier than that of wild type Under high salt stress,the root length and fresh weight of transgenic tobacco were increased by 62.33% and 52.21%,respectively,compared with wild type tobacco.Under the dehydration stress,the root length and fresh weight of transgenic tobacco were increased by 30.49% and 42.49% respectively compared with wild type tobacco.Under the low temperature stress,the root length and fresh weight of transgenic tobacco were 77.32% and 61.85% higher than that of wild type tobacco respectively.The transgenic tobacco showed enhanced cold resistance phenotype,including higher survival rate(1.65 times higher than that of wild type),lower electrolyte leakage,higher content of soluble sugar and proline(1.01 times and 0.31 times higher than that of wild type)Therefor,our results showed that overexpression of CsCBF2 in tobacco significantly enhanced many abiotic stress.3.Global transcriptome profiles of wild tobacco and transgenic lines L5 and L17 were compared using RNA-Seq technology.High-quality reads were used for a de novo assembly.In total,29,9333 transcripts,and 20,3218 Unigenes were achieved.The BLAST program was used for searched these unigenes against seven public databases,including SwissProt,Nr,Nt,KEGG,interproscan,KOG,GO.45428(22.35%),79981(39.36%),150753(74.18%),39584(19.48%),112787(55.50%),32342(15.91%),and14498(7.13%)unigenes were annotated in these databases,respectively.4.DESeq R program was used to obtain DEGs.In WT vs L5,2613 DEGs were obtained,among which 1654 DEGs were up-regulated,and 959 down-regulated;in WT vs L17,2545 DEGs were obtained,among which 1762 DEGs were up-regulated,and 783down-regulated;763 DGEs were shared by WT vs L5 and WT vs L17.Transcriptome profiles of both transgenic lines were very similar,but transcriptome profiles of transgenic lines were very different form that of wild tobacco.5.In these 763 DGEs,33 ERF transcriptional factor were obtained,among which 32 ERF were up-regulated;22 WRK transcription factors were found,and all of them were up-regulated;MYB transcriptional factors were obtained,among which 8 MYB were up-regulated and 2 transcription factors down-reglated;6 DREB were found,and all of them were up-regulated;all 6 NAC in these DGEs were up-regulated.6.GO enrichment analysis of DEGs were performed,and it was found that the metabolic process,cell process and cell metabolism were enriched in the biological process;the cell fraction,cell and cell membrane were significantly enriched in the cell fraction;protein binding,enzyme activity,transcriptional regulator activity were enriched in the molecular function.KEGG enrichment analysis of DEGs were implemented,and it was found that metabolic pathway,signal transduction pathway,unsaturated fatty acid biosynthetic pathway,amino acid synthesis pathway were enriched significantly.7.Combined with physiological,biochemical,andtranscriptome analysis,overexpression of CsCBF2 enhanced stress resistance by regulating the expression of related genes involved in ethylene pathway,signal transduction pathway,unsaturated fatty acid synthetic pathway,cell wall repair and defense regulation.