Cloning And Functional Analysis Of PtoABCG40 Gene In Populus Tomentosa

Poplar is a fast-growing,strong adaptable and widely distributed tree with important greening afforestation and industrial uses.However,poplar encounters biotic and abiotic stresses during their life cycle,such as pests and diseases,drought,cold,heat,salinity and chemical contamination of soil in the natural environments.These conditions can delay plant growth and development,restrict plant distribution,reduce productivity,and in extreme case,result in plant death.The effect of drought on the distribution and growth of poplar is a particularly serious issue.Therefore,the cultivation of fast-growing and drought-resistant poplar varieties is an issue that needs urgent attention.The introduction of new stress-tolerant genes into the poplar genome by transgenic technology is a very promising strategy.Abscisic acid is an important plant hormone that plays vital roles in plant response to biotic and abiotic stresses.When plants are adversely affected by low temperatures,drought,high salt,the level of ABA in plants will increase to activate the ABA signaling pathway and trigger a series of downstream biochemical and physiological responses,thereby adapting plants to adverse environments.When the plants are exposed to drought stress,the guard cells perceive the ABA signals will promote stomatal closure to reduce transpiration and reduce the loss of water to enhance drought tolerance.ATP-binding cassette(ABC)transporter is one of the largest family transporters in all kingdoms of life.ABCG is the largest subfamily,which plays very important roles in plant organ growth and development,cuticle formation,hormone transport,antibiotics transport,secondary metabolite secretion,response to biotic and abiotic stresses,and plant-microbe interaction.For example,Arabidopsis AtABCG25 and AtABCG40 are involved in regulating the transport of ABA to guard cells,causing stomatal closing and enhancing the drought tolerance in plants.At present,the research on drought tolerance of poplar mainly focuses on traditional cross breeding and enhancing the drought resistance of transgenic plants by introducing exogenous genes to enhance the lipid peroxidase protective enzyme system in plant cells.There are few reports on the activation of ABA signaling pathway to regulate the stomatal opening and closing of guard cells to enhance the drought resistance in poplar.Based on the function of Arabidopsis AtABCG40 in drought resistance,we attempted to clone similar genes in poplar and verify the function of the genes,and applied them to drought-tolerant breeding of poplar.In this study,the PtoABCG40gene was cloned from Populus tomentosa,and the plant overexpression vector was constructed and transferred into wild-type Arabidopsis thaliana and Populus tomentosa.The transgenic plants were tested for drought resistance.The detailed research results are as follows:(1)Cloning and bioinformatics analysis of PtoABCG40 geneThe PtoABCG40 gene cloned from Populus tomentosa cDNA was 4350 bp and encoding 1449 amino acid residues.Bioinformatics analysis showed that PtoABCG40belongs to PDR-type transporter with NBD-TMD-NBD-TMD structure.This protein has the closest relationship with ABCG proteins from Arabidopsis thaliana and Populus euphratica.We speculated that it may have a similar function to AtABCG40and is associated with the ABA signaling pathway that plays a key role in drought stress response.(2)Tissue expression and stress-induced expression analysis of PtoABCG40 geneTissue expression analysis by real-time PCR showed that PtoABCG40 was expressed in roots,stems,leaves and petioles,and the expression level in leaves was the highest.qPCR assay of PtoABCG40 after drought treatment showed that the its expression levels increased in all tissues.(3)Subcellular localization of PtoABCG40We constructed 35S:PtoABCG40-GFP expression vector and transiently expressed in tobacco leaves.It was found that PtoABCG40 was localized at the plasma membrane,endoplasmic reticulum and nucleus.(4)Drought tolerance analysis of transgenic plants overpressing PtoABCG40We constructed overexpressing vectors of PtoABCG40 and transformed wild type Arabidopsis and Populus tomentosa to obtain transgenic plants.Statistical analysis of the germination rate of WT and transgenie lines on 1/2MS medium containing 20%PEG.The results showed that the germination rate of transgenic Arabidopsis seeds was significantly lower than that of wild type.The root hairs of transgenic Arabidopsis seedlings were also significantly more than wild type under stress conditions.The transgenic Arabidopsis plants showed increased tolerance to drought stress comparing to wild type.(5)Analysis of the effect of PtoABCG40 gene overexpression on stomata opening and closingStomatal apertures were similar among wild type,mutant and transgenic lines under control conditions.However,stomatal apertures of the wild type and mutant plants were much greater than those of transgenic plants,in both opening and closing experiments when exposed to 50?M ABA.This indicated that the overexpression of PtoABCG40 in Arabidopsis can enhance the sensitivity of stomatal to ABA.(6)Analysis of K~+flow rate in guard cellsThe average flow rate of K~+in all material guard cells was negative before the10%PEG-8000 treatment.The mean of the K~+flow rates of all the materials was negative,indicating that the K~+influx occurred under normal conditions in guard cells.However,the average flow rate of K~+flow rate was positive after PEG-8000 treatment for 10minutes,indicating more K~+efflux in guard cells under PEG-8000 treatment conditions.The K~+mean flow rate in guard cells of transgenic Arabidopsis is significantly higher than wild type and mutant plants.The results showed that overexpression of PtoABCG40 could promote the efflux of K~+,leading to stomatal closure under the condition of PEG-8000 treatment.(7)Overexpression of PtoABCG40 gene promotes the transport of ABA in plantsBy counting the germination rates of wild-type and transgenic Arabidopsis seeds on 1/2 MS medium containing ABA,the germination rate of transgenic Arabidopsis was lower than that of wild type,which suggested PtoABCG40 promote more ABA uptake into seeds.Further treatment of Arabidopsis roots with ABA showed that the transgenic Arabidopsis plants had a higher absorption rate of ABA,which indicated PtoABCG40 facilitate ABA transport from root to shoot.In summary,this experiment preliminarily demonstrated that overexpression of PtoABCG40 in plants effectively increased the tolerance of plants to drought stress.Therefore,PtoABCG40 can be used as an excellent candidate gene to genetically improve the drought tolerance of plants,laying a foundation for us to cultivate new wide-adaptable drought-tolerant poplar varieties.