Study On The Regulation Of FT By Upland Cotton CO Protein

Flowering is a transition from the vegetative to the reproductive phase in plant life cycle,which is crucial for reproductive success and survival of higher plants.In recent years,Arabidopsis thaliana has been used as a model organism to study the regulation mechanism and signal transduction pathway of flowering time in higher plants.However,the key molecular mechanisms of flowering time in Gossypium hirsutum are still poorly understood.As an important cash crop,the flowering time of cotton will affect the reproductive growth period and fiber yield.Therefore,it is necessary to study the molecular mechanism of cotton flowering.While CO(CONSTANS)and FT(FLOWERING LOCUS T)are important regulators of flowering in many plants,the regulatory mechanism of the transcript factor of CO to the gene FT varies among different species.In Arabidopsis thaliana,CO promoted the expression of FT in leaves under long-day conditions,leading to flower transformation.On the contrary,Hd1(At CO homologous protein)inhibited Hd3a(At FT homologous gene)to delay rice heading under long day condition,and promoted rice heading under short day condition.In addition,G.hirsutum is an allotetraploid with A and D genomes.Both GhCO and GhFT have two parentals genomic homoeologs at the same time: GhCO-At and GhCO-Dt,and GhFT-At and GhFT-Dt.Therefore,the regulatory mechanism among these four genes is worthy of further analysis.Here,maxxa,a representative variety of G.hirsutum with good fiber quality,was used as the research material to explore the flowering regulation mechanism of Upland cotton by using the methods of PCR amplification and molecular cloning,real-time quantitative analysis,bioinformatic predication,as well as yeast one hybrid and tobacco transient transformation experiment.The main results are as follows:1.The 1.8 kb promoter sequences of GhFT-At and GhFT-Dt were obtained by PCR amplification and molecular cloning,and the corresponding coding sequences of1125 bp GhCO-At and GhCO-Dt genes were obtained.2.qRT-PCR analysis showed that GhCO and GhFT genes were both expressed in roots,stems,leaves,apical meristem,fibers and petals of G.hirsutum.Especially,GhCO and GhFT genes were highly expressed in leaves and apical meristem,suggesting that these genes may be invloved in the flowering regulation in G.hirsutum.Subsequently,the expression patterns of GhCO and GhFT genes in different developmental stages of leaves were analyzed.It was found that the expression of GhCO and GhFT genes in leaves increased with the development days of cotton regardless of short or long day conditions,but the increase amplitude in long day conditions was slower than that in short day conditions.In addition,we found that the flowering time of cotton was different under different light conditions,and the flowering date under short day condition was earlier.Therefore,it is speculated that the expression difference of GhCO and GhFT between long and short sunshine may be the main reason for the difference in flowering time of cotton under different light conditions.Combining the similar expression patterns and tissue specificity of GhCO and GhFT genes at different developmental stages in leaves,and the known functions of their homologs in Arabidopsis thaliana,we speculated that GhCO might be an upstream regulator of GhFT.3.Five possible CO binding elements on GhFT-At and GhFT-Dt promoters were predicted usingbioinformatics analysis.4.We performed yeast one-hybrid assay and showed that GhCO-At and GhCO-Dt could bind to two CO-binding elements on the promoters of GhFT-At.However,when these two elements were mutated,the binding ability was lost.5.Transcriptional activation assays showed that both GhCO-At and GhCO-Dt protein activated the expression of GhFT-At.These results indicated that the GhCO-At and GhCO-Dt may both be important positive flowering regulators in upland cotton,and the two proteins can interactively regulate GhFT-At,forming a more comlex activative regulation system than that in Arabidopsis.Our findings are of great significance for understanding the cotton flowering regulation network and improving the cotton flowering time and maturity cycle.