Cloning Of Phospholipase D?(CbPLD?) Gene In Chorispora Bungeana And Its Function Responding To Drought Stress

In the process of growth and development,plants often suffer from a variety of environmental stress factors,including drought,low temperature,salinity and heavy metals,which have a great impact on the growth of plants and the production of crops.Among the influencing factors,drought stress is particularly harmful to plants.One of the main manifestations of plant stress is the destruction of the plasma membrane,and membrane lipid degrading enzymes play an important role in this process.Phospholipase D(PLD)is a very important cellular phospholipid-metabolizing enzyme and plays an important role in signal transduction,stress defense responses,and seed germination.Abscisic acid(ABA),a plant hormone,also plays a crucial role in plant responses to cold,drought,and salt stress.In this study,PCR and RACE techniques were used to clone the phospholipase D?(Cb PLD?)gene from Chorispora bungeana,and the relative gene expression of the Cb PLD? gene in different tissues and stress conditions were studied by real-time quantitative PCR(RT-q PCR).In addition,the signal transduction relationship between PLD and ABA was also investigated under drought stress.The effects of ABA on the wild-type Arabidopsis thaliana(WT)and the konckout of PLD? under drought stress were also studied in model plant Arabidopsis thaliana.The effects of pld? lines on seed germination,membrane system and osmoregulatory substances.The results are as follows:(1)Phospholipase D?(Cb PLD?)(Genebank No.: MF951104)was cloned from Chorispora bungeana,and its structural composition was analyzed and predicted.The full-length c DNA of Cb PLD? was 2937 bp and the open reading frame(ORF)was2580 bp.The 5' and 3' untranslated regions(UTR)were 160 bp and 197 bp,respectively,and encoded 859 amino acid proteins with a molecular weight of 96.3KDa and an isoelectric point of 7.88.Secondary structure analysis predicts that Cb PLD? contains a "FIYIENQYF" domain and two HKD domains belonging to PLD family as their activity region.The putative Cb PLD? protein sequence contains a39.23% random coil,26.31% alpha helix,24.68% extended strand and the 9.78% beta turn.The alpha helices and random coils are the major secondary structure interlaces of the Cb PLD? protein.Phylogenetic analysis showed that Cb PLD? is highly identical to PLD from Arabidopsis thaliana PLD?.(2)The tissue-specific expression and the relative expression of Cb PLD? gene under 4°C low temperature,150 m M Na Cl,0.3 M Mannitol and ABA treatment were studied.The results showed that Cb PLD? gene was expressed in roots,stems and leaves in Chorispora bungeana,with the highest expression in roots and the lowest in leaves,indicating that Cb PLD? gene expression has tissue-specific,and Cb PLD? gene is widely involved in actively responding to low temperature,salt,drought and other abiotic stresses,in addition to plant hormone ABA can also induce Cb PLD? gene expression.(3)The relationship between PLD and ABA in Chorispora bungeana was studied under drought stress.The results showed that: the cell membrane of Chorispora bungeana was damage under drought stress,relative electrical conductivity and MDA content were increased;PLD and ABA will respond to drought stress,and the PLD activity and ABA content were increased under drought stress.Exogenously added PLD inhibitor 1-But,its ABA content decreased,and the ABA content increased after exogenously added PA,indicating that PLD/PA will promote the accumulation of ABA.Under drought stress,exogenous addition of different concentrations of ABA(10 ?M,50 ?M,100 ?M)promoted the PLD activity of Chorispora bungeana,and 100 ?M ABA had the most significant effect,but PLD activity was inhibited after ABA synthesis inhibitor sodium tungstate treatment.After co-treatment with sodium tungstate and Ca Cl2,the inhibitory effect of PLD activity was alleviated,indicating that ABA can promote PLD activity through Ca2+ and respond to drought stress.(4)The deletion of PLD? gene can inhibited the seed germination of Arabidopsis under drought stress.Exogenous ABA further inhibited the seed germination of pld? mutant under drought stress,indicating that both PLD? and ABA are involved in regulating the seed germination process of Arabidopsis thaliana under drought stress.In addition,PLD? gene and ABA interact with each other to regulate membrane stability and osmotic adjustment substances in Arabidopsis thaliana.