Identification And Functional Verification Of Cold Responsive Genes In Cotton

Cotton(Gossypium spp.)is the most important natural fiber crop in the world.Low temperature will reduce the germination rate of cotton seeds,and make the cotton plants be susceptible to diseases,which will eventually lead to a reduction in cotton production.Tetraploid cotton cultivars Gossypium hirsutum L.and Gossypium barbadense L.are the main cultivars.G.hirsutum has high yield and wide adaptability,but the fiber quality is average.G.barbadense has good quality of fiber,but poor resistance to stress,so the two have obvious complementary advantages.In this study,by comparing the phenotypes of G.hirsutum and G.barbadense seedlings under cold stress,we found that G.hirsutum has stronger cold stress tolerance than G.barbadense.Through transcriptome sequencing and differentially expressed gene analysis of leave tissue of G.hirsutum and G.barbadense under normal temperature(28 °C)and cold stress(4 °C)at different time interval,we identified the genes and metabolic pathways that differentially responsed to cold stress.In G.hirsutum and G.barbadense,8713 and 12461 differentially expressed genes(DEGs)under cold stress were identified,of which 3884 DEGs were shared by G.hirsutum and barbadense.Additionally,1518(28%)DEGs were special to G.hirsutum,and 3577(48%)DEGs were special to the G.barbadense,indicating the consistency and specificity of response to cold stress in G.barbadense and G.hirsutum.According to orthologous genes between G.hirsutum and G.barbadense,we found that 7401 and 10285 orthologous genes were induced under cold stress in G.hirsutum and G.barbadense,respectivly.Moreover,814 and 3577 genes were specially induced under cold stress in G.hirsutum and G.barbadense,respectively.By GO enrichment and KEGG enrichment analysis,it was found that the DEGs in G.barbadense were mainly enriched to embryonic development,while the special DEGs in G.hirsutum was specifically enriched to photosynthesis,circadian rhythm and other pathways.We found that the fatty acid pathways were very different in G.hirsutum and G.barbadense,including ACCase,FAS,SAD,FAD and LACS family genes,17 DEGs were induced in G.hirsutum and 24 DEGs were induced in G.barbadense.At the same time,most of the expression level of ACCase genes were reduced in G.barbadense.Three genes of b HLH family were up-regulated after 1hour under cold stress in G.hirsutum.PIFs are subfamily members of the b HLH,which play a key role in temperature and light signal transduction.Silencing Gh PIF8 by VIGS can significantly reduce the cold tolerance of G.hirsutum,indicating that Gh PIF8 is positive to G.hirsutum’s resistance to cold.In addition,we detected 1338 and 1914 transcription factors(TFs)induced by cold stress in G.hirsutum and G.barbadense,respectively.With the extension of the treatment time,the number of TFs responsed to cold stress showed a gradual upward trend.After 1 hour of cold stress treatment,25 TFs were differentially expressed in G.hirsutum and 15 TFs were differentially expressed in G.barbadense,indicating that G.hirsutum respond to the cold stress more quickly.