Study On The Cold Tolerance Evaluation And Molecular Expression In Tobacco Seedling Stage

Cold stress is a major environmental factor that affects plant growth,development,productivity and distribution.Different genotypes of tobacco have different tolerance to cold,but the knowledge of their molecular mechanismis still limited.In the present study,a batch of tobacco germplasm resources resistant to cold stress were identified,and comparative genome-wide transcriptome and proteome analysis on two tobacco(Nicotiana tobacum L.)cultivars,cold-tolerant cultivar NC567 and cold-sensitive cultivar Taiyan8,was performed using RNA-seq and iTRAQ technology.The results are as follows:(1)A total of 424 tobacco resource materials were selected.Using the identification method of cold tolerance at the seedling stage,15 cold-resistant materials including NC567,and 72 medium cold resistant materials including K326,and 337 cold sensitive materials includingTaiyan 8 were screened out.(2)The further genetic analysis showed that there is the significant genotype-dependent tolerance to cold stress in tobacco,and the cold tolerance ability can be inherited,which exhibits the heterosis over parents.(3)Comparative genome-wide transcriptome analysis on two tobacco(Nicotiana tobacum L.)cultivars,cold-tolerant NC567 and cold-sensitive Taiyan8,was performed using RNA-seq technology.After the first assembly,total length of unigenes is from 101,308,644 to 123,781,795 bp,the N50 length is from 1357 to 1475 bp,and 152,688 unigenes in NC567 and 144,160 unigenes in Taiyan8 were identified,respectively.Functional classification of cold-responsive(COR)genes showed that the genes involved in cell wall metabolism,transcription factors,ubiquitinproteasome system(UPS)and signaling are over-represented,and the COR genes are specifically induced during cold stress in NC567.Pathway analysis revealed the significant enrichment of the COR genes in plant circadian clock.(4)NC567 and Taiyan8,two cultivars with different sensitivities to low temperature,were used in Isobaric Tags for Relative and Absolute Quantitation(iTRAQ)based proteomics to uncover their different mechanisms in response to cold stress.A total of 4315 distinct proteins were identified and the differential proteins in four comparison sets were used for further analysis.The Gene ontology(GO)analysis indicated that the majority of differentially expressed proteins were in the categories involved in metabolic and cellular processes.Surprisingly,there were 55 proteins decreased in NC567 but increased in Taiyan8 in response to cold,while the levels of 42 proteins were lower in Taiyan8 than NC567 at normal temperature but higher in Taiyan8 than NC567 under cold treatment,suggesting different responses to cold stress in these cultivars.The levels of polypeptides involved in protein synthesis and degradation,photosynthesis and respiration,as well as ROS scavenging,were different in the comparison sets,implying that protein and energy metabolisms may be important for the establishment of cellular environment at low temperature.