Identification Of Differentially Expressed Genes (DEGs) In Response To Abiotic Stresses In Brassica Napus L. By CDNA-AFLP Technique

Brassica napus L.is a very important edible oil crop in China.As the largest producer of rape in the word,China still faces a severe challenge in the production and development of rapeseed industry.China’s rape cultivation industry is mainly concentrated in the middle and lower reaches of the Yangtze River.The adverse environmental impacts such as seasonal drought and even freezing damage occur frequently in the area,and can result in a significant reduction in the production of rape.The most effective way to reduce the losses caused by these adverse environmental impacts will be the breeding of resistant varieties containing various stress resistance genes,especially those that can simultaneously respond to several different stress conditions.Identification of the abiotic stress-induced genes at the transcriptional level would be an effective method to target stress response genes and explore the molecular mechanism of resistance to abiotic stresses.In this study,"Zhongshuang 11" was used as experimental material to analyze the differentially expressed genes(DEGs)in young leaves after extreme temperature duress(4℃ and 40℃)by using cDNA amplified fragments length polymorphism(cDNA-AFLP)technology.The DEG fragments were sequenced and the corresponding genes were identified by BLAST analyses against the public sequence databases.For 36 of these DEGs,specific quantitative PCR primers were designed,and semi-quantitative RT-PCR analysis was performed to further examine their expression patterns in response to salt stress,drought stress and water flooding stresses.Meanwhile,two genes that were highly up-regulated by high temperature stress were cloned and transformed into Arabidopsis thaliana for their overexpression analysis..The main results are resumed as follows:1.Analysis of differentially expressed genes in young leaf of B.napus under extreme temperature stressAccording to cDNA-AFLP analysis,a total of 9728Transcript-derived fragments(TDFs)were amplified by using 256 primers combinations.The number of fragments varied from 10 to 55 with an average of 38 fragments per primer combination.The size of fragments varied from 50 to 700 bp.Although the majority of bands showed no change in intensity between control and extreme temperature stressed samples,we detected 543 differentially expressed gene(DEG)fragments,of which 361 were responsive to heat stress,328 were responsive to cold stress,and 104 were responsive to both heat and cold stresses..Analysis of sequencing data of these 585 DEG fragments allowed the identification of 538 non-redundant sequences,of which 445 can match known gene and/or protein sequences,while the remaining 93 cannot find their homologous sequences from the public databases of actual version.The Gene Ontology(GO)enrichment analysis of 445 DEGs showed that 102(22.92%)encode proteins with enzyme regulator activity;45(10.11%)encode proteins with ATP binding function;34(7.64%)encode proteins with protein binding property;25(5.62%)encode proteins with kinase activity;25(5.62%)DEGs encode proteins with transcription factor activity;23(5.17%)encode proteins with metal binding property;19(4.277%)encode proteins with transport activity;14(3.15%)encode proteins with ubiquitin protein ligase activity.Other types of DEGs(with proportions<3%)that encode proteins with activities of DNA binding,RNA binding,zinc binding,calcium binding,nucleic acid binding,GTP binding,structural molecular,signal transduction,carbohydrate binding etc,were also identified.96(21.57%)DEGs encode proteins with unkwown functions.Interestingly,10 DEGs were identified as encoding heat shock proteins,and 2 DEGs as encoding low temperature induced proteins.Some of these DEGmembers encode proteins with multi-funtions.Subcellular localization analysis showed that 85(19.10%)DEGs are located in nucleus,61(13.71%)located in chloroplast,48(10.79%)located in cell membrane,45(10.11%)located in cytosol,44(9.89%)located in the cytoplasm,27(6.07%)DEGs located in extracellular compartment,26(5.84%)localized in mitochondria,24(5.39%)localized in chloroplast,19(4.27%)located in cell wall,15(3.37%)located in thylakoid membrane.Other types of DEGs(with proportions<3%)were predicted to be localized in vacuolar membrane,Golgi apparatus,endoplasmic reticulum,extra body and photosystem II.158(35.51%)DEGs cannot be localized in a subcellular region with the used prediction software.Some of these DEGs were predicted to be localized in multi-compartiments in the cell.2.Analysis of expression patterns of 36 DEGs in leaves of B.napus under salt stress,drought stress and water stress.36 DEGs were selected from the total identified DEGs based on their good quality during the cDNA-AFLP analysis.Their expression patterns in the leaves of "zhongshang 11”under salt,drought and water stresses in comparison with the controls were analysed by semi-quantitative RT-PCR technique.The results showed that three genes were responsive to both extreme temperature and salt stresses;one gene was responsive to both extreme temperature and water logging stresses;three genes were responsive to extreme temperature、salt and drought stresses at the time;two gene were responive to extreme temperature、salt、drought stress and water logging stress at the same time.3.Construction of overexpression vector of 2 B.napus DEGs and transformation of Arabidopsis thalianaTwo whole length DEGs corresponding to TDF-4 and TDF-9 were amplified from the leaf cDNA of "Zhongshuang 11",and successfully cloned into the overexpression vector pCXSN.The two constructions were successfully transformed into A.thaliana line colO,and we obtained 12 and 15 transformed T0 plants for TDF-4 and TDF-9,respectively A.thaliana.Analysis of a few T2 plants showed that these transformed plants were A.thaliana highly sensitive to high temperature,and became completely wilted after 24h exposure to 40 ℃.