Construction Of UCPs-overexpressing Vectors And Functional Analysis Of Arabidopsis UCP2 And UCP6 During Seed Germination

Mitochondrial uncoupling proteins(UCP,Uncoupling proteins)are called the?proton electrochemical potential?energy dissipation pathway because they can release the transmembrane H~+flux and uncouples oxidation and phosphorylation and dissipates chemical energy into metabolic heat.UCPs are encoded by nuclear genes and localized to mitochondrial membranes.Many experiments have proven that UCPs are involved in processes of stress adaptation and development in plants,but the physiological and molecular mechanisms are not clear.In order to elucidate the roles of UCPs in the processes of plant growth and stress responses from molecular and genetic aspects,we generated the UCP overexpression and GUS marker lines.Meanwhile,we also screened for homozygous T-DNA insertion mutants of some UCP family members.Furthermore,the insertion mutants of ucp2 and ucp6 were used as the materials to explore the roles of UCPs during seed germination in Arabidopsis thaliana.The main results are as follows:1.Cloning the CDS region of the UCP genes(UCP1:At3g54110;UCP2:At5g58970;UCP4:At4g24570;UCP6:At5g09470).The cloned fragment of each gene was successfully transferred into the overexpression vector p GWB2 through Gateway technology and the UCP-overerexpressing plants were generated.The expression level of three UCP1 overexpression lines(UCP1-OE)was increased by 5.24 fold,1.87 fold,and 6.47 fold;the expression level of three UCP2-OE lines was increased by 2.17 fold,3.92 fold,and 4.00 fold;the expression level of three UCP4-OE lines was increased by 5.65 fold,2.57 fold,and 6.57 fold;the expression level of three UCP6-OE lines was increased by 4.85 fold,4.65 fold and 4.11 fold.2.Cloning UCP2 and UCP6 promoter regions.The cloned fragments were successfully introduced into the GUS fusion vector p GWB3 through Gateway technology and the transgenic lines were generated.3.The expression pattern of UCPs were analyzed by quantitative real-time PCR(q RT-PCR):UCP1 is highly expressed in roots,stems,leaves,flowers,and seeds.UCP2 is mainly expressed in flowers.UCP2 and UCP6 are expressed in young seeds;UCP4 is mainly expressed in leaves.The GUS staining of UCP2pro::GUS and UCP6pro::GUS further confirmed the expression patterns.UCP6 is highly expressed in seeds and pistils,but no expression in anthers,which is different from the expression pattern of UCP2.4.Identifying the T-DNA mutants.We ordered mutant seeds from ABRC.After genotyping and RT-PCR analysis,we confirmed that the homozygous ucp2(Salk?037074,Salk?080118,and Salk?040242)and ucp6(Salk?111403)are null mutants.Then we used q RT-PCR to further examine whether UCP2 is responsive to some abiotic stresses.The results showed that UCP2 is more sensitive to cold stress,and the expression level was increased by 1.76 times at 4 h,and 1.85times at 24 h.In addition,UCP2 is also responsive to mannitol,salt,and ABA stress,suggesting that UCP2 plays an important role in the response to stresses in Arabidopsis.5.Investigating the roles of UCP2 and UCP6 during seed germination.The results indicate that the seed germination of ucp2 and ucp6 was delayed compared to the wildtype.The germination of ucp2 and ucp6 lines was inhibited by salt and mannitol stress.In the medium containing different concentrations of ABA,it was found that the germination of ucp2 and ucp6was delayed.At the fourth day of 0.3?M ABA treatment,the germination rate of wildtype seeds reached 78.7%,but the germination rate for ucp2 and ucp6 was only about 15.2%and 9.6%,respectively.The greening rate of ucp2 and ucp6 was also affected.When Col-0 green rate reached80.0%,the seedlings of ucp2 and ucp6 was not unable to turn green.ABA has no influence on seedling development of ucp2 and ucp6.In addition,different concentrations of salt and mannitol delayed the germination of ucp2 and ucp6 to different degrees.6.During the process of seed germination,we supplied mannitol,salt,ABA and Paclobutrazol(PAC,the inhibitor of gibberellins)and then stained the transgenic lines UCP2pro::GUS and UCP6pro::GUS.The results showed that the expression of UCP2 and UCP6had a sharp increase during seed germination,further indicating that UCP2 and UCP6 may play an important role in seed germination.The results of q RT-PCR showed that the expression of three negative transcription factors of seed germination,ABI3,ABI4 and ABI5,was increased by ABA treatment in ucp2 and ucp6,indicating that ucp2 and ucp6 are sensitive to ABA during germination because of ABI3,ABI4 and ABI5 implementation.In summary,we generated the UCP overexpression lines and the GUS marker lines.T-DNA insertion mutants of UCP2 and UCP6 on were explored.The expression of ABI3,ABI4 and ABI5was up-regulated in ucp2 and ucp6 in the process of germination,which may be the main contributing factor leading to the delayed germination of ucp2 and ucp6 under ABA stress.The results provide a basis for further study of the functional and molecular mechanisms of UCPs in plant growth and stress adaptation.