Study On Key Amino Acid Sites Of AtGDPD-Like3 In Arabidopsis Root Hair Development Gene

Root hair is a tubular structure formed by the outward protrusion of single cells in the plant's root epidermis.Root hair,which accounts for 77%of root surface area,promotes the uptake of water and nutrients by increasing the absorption area of epidermal cells,and realizes plant fixation and environmental interaction.Root hair development is an extreme type of cell polarity growth apical growth,which can grow more than ten times the length of cells in a specific direction within a dozen hours.This apical growth is a complex process,which is regulated by many factors,such as calcium concentration,inositol phosphate,vesicle transport,reactive oxygen species,cell wall,cytoskeleton and so on.AtGDPD-Like is a similar gene encoding glycerophosphoryl diester phosphodiesterase?GDPD?,the family gene AtGDPD-Like3 was highly expressed in root hair tissue of Arabidopsis thaliana.The expression of this gene is closely related to the morphology of root hairs in Arabidopsis thaliana and is necessary for root hairs elongation,the shv3 deletion mutant leads to serious root hairs development defects.In order to study the mechanism of the function of AtGDPD-Like3 protein,the key sites in the amino acid sequence of AtGDPD-Like3 protein were screened and identified.The main results are as follows:A T-DNA insertion mutant SALK₁37845 of AtGDPD-Like3 was identified by the three-primer method,semi-quantitative RT-PCR results showed that SALK₁37845 is a knockout mutant of AtGDPDL-Like3.The mutant atgdpdl3 has serious root hair development defects,and its 28-day seedlings have a slow growth phenotype.The Locus Name of AtGDPD-Like3 is At4G26690 containing 8 exons and 7 introns.The full-length sequence of CDS consists of 2280 nucleotides that encode 759 amino acids.Homology search showed that the homologous genes of the AtGDPD-Like3 exist in various plants such as Glycine max,Populus trichocarpa,Sorghum bicolor and other species.By sequence alignment analysis,nine conserved amino acid sites,Asp86,Asp128,Ser206,Gly261,His289,Asp400,Ser538,Va1556 and Asp628,were selected as the target sites for the mutation.In order to identify the key amino acid sites of AtGDPD-Like3,a series of single-point mutations of AtGDPD-Like3 with different single-point mutations were constructed according to nine conserved amino acid sites of AtGDPD-Like3 protein,and then transferred to mutant strains of atgdpdl3 for functional complementation experiments.After observing the degree of root hairs defect of atgdpdl3 restored by single point mutation AtGDPD-Like3 and quantifying the root hairs,it was found that the conserative mutations at both His289 and Asp86 in AtGDPD-like3 could not restore the root hair development defect phenotype of atgdpdl3 mutant,suggesting that the function of the AtGDPD-Like3 protein was lost after the mutations of Asp86 and His289,Asp86 and His289 are key amino acid sites affecting AtGDPD-Like3 function;the conservative mutations at Ser206,Val556 and Asp628 of AtGDPD-Like3 protein could obviously restore the phenotype of abnormal root hair growth in the mutant of aigdpdl3,suggesting that the mutations of Ser206,Val556 and Asp628 have no decisive effect on the complementary function of AtGDPD-Like3,Ser206,Val556,and Asp628 are not key amino acid sites that affect the function of AtGDPD-Like3 protein;the conservative mutations of Asp128,Gly261,Asp400 and Ser538 of AtGDPD-Like3 only partially restored the phenotype of root hair defect in the mutant of atgdpd1B,suggesting that the mutations of Asp128,Gly261,Asp400 and Ser538 also had certain effects on the function of AtGDPD-Like3,Asp128,Gly261,Asp400 and Ser538 were also the key amino acid sites affecting the function of AtGDPD-Like3 protein.In conclusion,Asp86,His289,Asp128,Gly261,Asp400 and Ser538 are key sites in the amino acid sequence of AtGDPD-Like3 protein to determine its function;the conserved amino acid residues of Ser206,Val556 and Asp628 are not the key sites affecting the function of AtGDPD-Like3 protein.