Cloning Of Chorispora Bungeana PLDζ Gene And Expression Analysis Of Three PLD Genes

In the process of plant growth,its growth and development are seriously affected by biotic stress and abiotic stress.In recent years,along with the global warming,the climate changes frequently,the pollution is serious,and the soil is destroyed.Among many environmental factors,abiotic stress could affect plants in many aspects.For example,destroy plant plasma membrane,alter plant phenotype,change the content of plant endogenous hormones,affect plant photosynthesis,and promote stomatal closure and so on.Phospholipase D(PLD)is an extremely important transmembrane signal transduction enzyme and cellular phospholipid metabolism enzyme,which plays an important role in the process of plant growth and development.In this study,a new PLD gene from Chorispora Bungeana,named CbPLDζ,was cloned and analyzed by using RT-RCR,RACE,and real-time fluorescence quantitative PCR(RT-q PCR).The relative expression of CbPLDζ gene under different tissues and stress conditions was studied;in addition,the functions of CbPLDγ and CbPLDδ genes were also discussed.The findings are as follows:(1)The CbPLDζ gene was cloned from Chorispora Bungeana,and it was found that the CbPLDζ gene has a full length of 3147 bp,an open reading frame(ORF)of2197 bp,and encodes 989 amino acids.α-helix and random curl are the main secondary structural interlaced domains of the CbPLDζ protein.Phylogenetic analysis showed that CbPLDζ and Arabidopsis thaliana PLDζ genes were most closely related.(2)The tissue-specific expression of CbPLDζ gene and its relative expression levels under 150 m M Na Cl,0.3 M mannitol and 100 m M ABA were studied.The results showed that the CbPLDζ gene in Chorispora Bungeana were expressed in the roots,stems and leaves,but the expression level is different.This indicated that the CbPLDζgene expression is tissue-specific.The CbPLDζ gene actively responded to a variety of abiotic stress,and the plant hormone ABA also induced the expression of CbPLDζ gene.(3)The subcellular localization vectors p MDC83-CbPLDγ-GFP and p MDC83-CbPLDδ-GFP were constructed for the CbPLDγ and CbPLDδ genes of Chorispora Bungeana,and the recombinant vectors were transferred into tobacco for transient expression.The subcellular localization of the genes was observed using a confocal microscope and it was found that CbPLDγ and CbPLDδ proteins are all significantly accumulated on the cell membrane.(4)The promoter fragments of CbPLDγ and CbPLDδ genes from Chorispora Bungeana were cloned by "chromosome walking method",and their functions were predicted.The tissue localization GUS expression vectors p BIB-GUS-CbPLDγ-Pro and p BIB-GUS-CbPLDδ-Pro for the CbPLDγ and CbPLDδ genes of Chorispora Bungeana were constructed,and the recombinant vectors were transferred into Arabidopsis.After stable expression,GUS staining revealed CbPLDγ.CbPLDδ gene is expressed in roots,hypocotyls,leaves and flowers.CbPLDγ gene is enriched in anthers and root tips,but less expressed in hypocotyls.CbPLDδ gene was significantly enriched in root tips and anthers,and less expressed in petals.(5)Agrobacterium-mediated Arabidopsis inflorescence dip method was used to transfer the constructed overexpression vectors p MDC83-CbPLDγ and p MDC83-CbPLDδ into Arabidopsis.The fourth generation homozygotes were screened by basta,and RT-PCR and RT-q PCR method was used to identify the fourth generation homozygotes,and the homozygous seedlings of two lines were selected for the function exploration under drought stress.It was found that under drought stress,compared with wild-type plants,CbPLDγ and CbPLDδ gene transgenic lines had lower membrane damage,higher osmotic adjustment substance content,lower hydrogen peroxide content,and stronger antioxidant enzyme activity.It can be explained that the drought tolerance of transgenic lines is enhanced.