Molecular Mechanism Study Of Flowering Time Regulation By Three Circadian Clock Genes In Gossypium Hirsutum

Cotton is the most important fiber crop in the world,and it occupies a pivotal position in our country's national economy.In recent years,due to the low degree of mechanization,rising labor costs and competition for land between grain and cotton,the area of cotton planted in the Yellow River and Yangtze River basins has been drastically reduced.Early-maturing cotton is an important type of cotton variety,with a short growth period and concentrated boll-opening date.Breeding more early-maturing cotton varieties has become an important method to solve the current dilemma of cotton planting.The growth period and boll-opening date of cotton mainly depend on its flowering time,but there are few reports on the molecular mechanism of cotton flowering time regulation,and Only a few flowering genes have been identified.The photoperiod is the most reliable external signal.Plants judge whether the external environment is suitable for flowering by sensing the photoperiod.The circadian clock plays an important role in the photoperiod flowering pathway,but the roles of circadian clock genes in regulating the flowering time of cotton are still unknown.LUX,ELF3 and CCA1 are the important components of the plant circadian clock.They are involved in regulating multiple other circadian clock genes and genes in the output pathways,and perform functions of regulating flowering time in many plant species.Therefore,in this study,homologous genes of LUX,ELF3 and CCA1 were identified in 27 plant species,and their physicochemical properties and conservation were analyzed.By analyzing the different expression of GhLUX1,GhELF3 and GhCCA1 in the early-and late-maturing upland cotton varieties,and overexpressing them in Arabidopsis and silencing them in cotton,their functions in regulating flowering time were determined.Finally,the interaction between the three genes and their regulation on flowering genes were preliminarily explored.The main conclusions are as follows:1.Fourty two LUXs,fifty eight ELF3 s and fourty CCA1 s were identified in 27 plant species.All LUXs,ELF3 s and CCA1 s are hydrophilic proteins;and the isoelectric points of all LUXs are greater than 7.5,while the isoelectric points of most ELF3 s are less than 7.5,indicating that LUXs and ELF3 s have opposite charges in plant cells,which provides the necessary physiochemical conditions for their possible interactions in plant cells.The results of phylogenetic analysis show that LUXs and CCA1 s in the species belonging to the same Orders and Families are divided into the same branch,while ELF3 s in the dicots are divided into two branches.The exon-intron structures of LUXs,ELF3 s and CCA1 s are highly conservative.LUXs and CCA1 s contain longer conserved motifs and a Myb DNA-binding domain,but ELF3 s contain shorter conserved motifs and do not contain any known domains.In addition,the distribution of conserved motifs in LUXs and CCA1 s promoters is more conservative than the distribution of conserved motifs in ELF3 s promoters.In upland cotton,Arabidopsis and rice,the circadian expression rhythms of LUXs and CCA1 s are more conservative than those of ELF3 s.These results indicate that during plant evolution,the functions of LUXs and CCA1 s may be conserved,while the functions of ELF3 s may be diverse.2.Under long day(LD),short day(SD),constant light(LL),and constant dark(DD)photoperiod conditions,Gh FT and its upstream regulators,GhCOL1 and GhTEM1,show diurnal expression rhythms in both early-maturing upland cotton variety Z50 and late-maturing upland cotton variety GX11,indicating that the expression of flowering genes in cotton is under the control of the circadian clock.Under LL and LD,the expression levels of GhLUX1,GhELF3 and GhCCA1 are higher in Z50(except GhCCA1 under LD);while under SD and DD,the expression levels of GhLUX1,GhELF3 and GhCCA1 are higher in GX11.These results indicate that under long day conditions,late-maturing upland cotton varieties may have stronger circadian clock oscillations,while under short day conditions,early-maturing upland cotton varieties may have stronger circadian clock oscillations.Z50 plants in the dark were treated with continuous light at different times.It was found that light can reset the expression rhythms of GhLUX1,GhELF3 and GhCCA1.However,when light is started at different times,the reset oscillation rhythms of the circadian clock are different,which may lead to the different expression patterns of Gh FT,GhCOL1 and GhTEM1.3.Arabidopsis plants overexpressing GhLUX1,GhELF3 and GhCCA1 have late flowering times,increased rosette leaves,and late bolting times.Overexpression of GhLUX1,GhELF3 and GhCCA1 in Arabidopsis changes the expression of the circadian clock genes,including At LUX,At ELF3,At ELF4,At CCA1,At LHY and At PRR7.In the overexpression lines of the three genes,the expression of At FT is greatly reduced,which results in their delayed flowering.The expression changes of At FT upstream regulators,At CO,At TEM1,At GI and At FKF1,may be the reason for the reduced expression of At FT.Silencing GhLUX1 and silencing GhELF3 in cotton via VIGS(Virus-Induced Gene Silencing)lead to early flowering,while silencing GhCCA1 in cotton via VIGS leads to late flowering.After any one of the three genes is silenced,the expression of the other two genes is also up-regulated or down-regulated,indicating the reciprocal regulation between the circadian clock genes.In addition,after silencing GhLUX1,GhELF3 and GhCCA1 via VIGS,the expression of Gh FT,GhCOL1 and GhTEM1 is also changed correspondingly,which may lead to changes in cotton flowering time.4.The results of subcellular localization analysis showed that GhLUX1,GhELF3 and GhCCA1 are located in the nucleus,indicating that they perform functions in the nucleus.The results of transcriptional activation experiments showed that GhLUX1 has transcriptional activation activity in yeast,while GhELF3 and GhCCA1 don't have.GhLUX1 was divided into two parts,GhLUX1-N(1-154 Amino Acids,AAs)and GhLUX1-C(155-337 AAs),and it was found that GhLUX1-N has transcriptional activation activity.Yeast two-hybrid experiment showed that GhLUX1-C can interact with GhELF3,GhELF3-N(1-460 AAs),and GhELF3-C(467-705 AAs),indicating that GhLUX1 may be sandwiched between GhELF3-N and GhELF3-C;Bimolecular fluorescent complimentary experiment showed that the interaction of GhLUX1 and GhELF3 occurs in the nucleus,indicating that GhLUX1 may recruit GhELF3 to the promoters of downstream genes to regulate their expression.The results of yeast-one hybrid experiment and dual luciferase experiment showed that GhCCA1 can bind to the evening elements(AAATATCT)on the promoters of GhLUX1 and GhTEM1 and suppress transcription.