Function Characterization Of GaTOP6B, A Cotton Gene Regulate Endoreduplication, In Tolerance To Drought Stress

During the growth of a plant,the replication of the genome does not undergo mitosis resulting in an increase in the ploidy level of the cells is called endoreduplication,which plays an important role in development and physiology through cellular,metabolic and genetic effects.Drought is deemed as one of adverse conditions that could cause substantial reductions in crop yields worldwide.Cotton is much more taken as a pioneer plant adapted itself to drought stress,which provides us as an excellent model to characterize potentially new genes regulating drought tolerance in plants.In our present work,GaTOP6 B,encoding DNA topoisomerase Ⅵ subunit B,was identified and functionally characterized.The virus-induced gene silencing(ⅥGS)and over-expression(OE)were used to investigate the biological function of GaTOP6 B in Gossypium arboretum and Arabidopsis under drought stress.1.A homolog of TOP6 B in cotton,designated GaTOP6 B,was identified.The opening reading frame(ORF)of GaTOP6 B is 2010 bp,which encodes for a protein containing 669-amino acid.The amino acid sequence of GaTOP6 B was predicted to contain conserved GHKL,H2 TH,Transducer domains and shared high similarity with T.cacao and C.olitorius,which indicates that they might have originated from the same common ancestor.2.GaTOP6 B was constitutively expressed,at relatively higher levels in cotton leaves.The subcellular localization of GaTOP6 B protein showed that GaTOP6 B was specifically targeted to nucleus.3.The GaTOP6B-silencing plants generally exhibited a dwarf phenotype,a decreased ploidy level and reduced nuclear size.After subjected to water deficit for 20 days,Ga TOP6B-silencing plants grew much more wilted and reduced activity of antioxidant enzymes and decreased proline and chlorophyll content.The stomatal size and opening rate were markedly increased which might cause an accelerated rate of transpiration under drought stress,as the leaf RWC was much lower than that in WT.Several genes related to stress were remarkably repressed in the silencing plants such as RD29,NCED3,COR15 A,DREB1A.4.Ectopic overexpression of GaTOP6 B displayed a prompt growth compared with the wild-type.Flow cytometry analysis showed that an enhanced level of ploidy and the nuclear volume in the leaf of transgenic lines was obviously enlarged.The transgenic lines generally displayed an improved growth status,and the leaf RWC and proline content was relatively higher,the stomatal opening was much reduced and accompanied by a lower WLR in contrast with wild-type under the water-deficit condition.Stress-responsive genes including RD29 A,COR15A,NCED3 and DREB1 A were highly up-regulated in the transgenic plants than that in the wild-type under the water deficit condition.5.Transcriptome analysis of GaTOP6B-overexpressing Arabidopsis showed that genes in transgenic Arabidopsis had a high transcription level.Under 200 Mm mannitol treatment,DEGs of transgenic Arabidopsis enriched in metabolism that encode proteins and probably function in stress tolerance include plant defensin,cell well biogenesis,trehalose biosynthesis and glutathione metabolism,which were probably involved in eliciting physiological,biochemical and molecular changes at the cellular level,including enhanced defense,cell wall thickening,osmotic adjustment and scavenging ROS.