The Hormone Mechanism Of DPC Promoting Lateral Root Formation In Cotton Seedling And The Preparation Of Conjugated ABA ScFv

N,N-Dimethylpiperidinium choride(DPC)is a widely-used plant growth regulator in cotton(Gossypium hirsutum L.)production to increase lateral root formation.The degree of root branching influences the efficiency of water uptake and to cope with various abiotic stresses.In the study,with cotton seeds were soaking DPC solution,to explore the hormone mechanism of DPC promotes lateral root development.The main results were as following:1.LRP,LR number was increased by 32.1%,34.9%,21.8%and 10.9%on 4-7 d in DPC-treated cotton seedlings.And LR initiation occurred earlier in DPC-treated cotton seedlings by paraffin section.2.IAA concentration significantly increased in the root,but IAA and IAA conjugates content markedly decreased in the cotyledon on 2 and 3 d after treatment.A higher accumulation of IAA in the stele of DPC-treated cotton seedlings was observed in the 0.3-1.5cm behind the root tip,but unchanged in the cortex and epidermis.3 Expression of IAA conjugates hydrolase genes GhILR1,GhIAR3.1 and GhIAR3.2,and IAA efflux carrier genes GhPINl,GhPIN1b,GhPIN1c and GhPIN2 were up-regulated in DPC-treated seedling.While,expression of IAA biosynthesis genes GhTAAl,GhTAR2 showed no obvious affect and IAA influx gene GhAUX1 showed no obvious change after treatment.These results suggested that DPC induced endogenous IAA in LR priming region was mainly due to hydrolyzing IAA-amino acid conjugates to free IAA in cotyledon and then transporting IAA to this area.4.GA-mediated repressive effect on root development resulted from a dramatic reduction of LRP number and LRP density.GA content was decreased in cotyledon and root.Expression of GA bio synthesis genes GhCPS,GhKS,Gh20oxl and Gh20ox2 was down-regulated,while expression of GA metabolism genes Gh2ox1and Gh2ox2 was up-regulated.5 The GST-GhIAR3.1 fusion which was successful expressed in the supernatant of Escherichia coli.GST-GhIAR3.1 could cleave both IAA-Ala and IAA-Asp in vitro,which have not been previously reported in cotton.Vmax and Km values of GST-GhIAR3.1 fusion on IAA-Ala were 17.2 ?mol/min/mg and 320.67 ?M.6 GST protein and GhIAR3.1 were separated by Thrombin digest.The rabbit polyclonal antisera and mouse monoclonal antibody against GhIAR3.1 were successfully achieved.And we establish a double sandwich ELISA by rabbit polyclonal antisera and mouse monoclonal antibody.The GhIAR3.1 content increased by 17.8%in cotyledons on 2 d after treatment with double sandwich ELISA.In this paper,we also studied the binding characterization of scFv antibody S12B8 with abscisic acid(ABA)and conjugated ABA by competitive ELISA and in silico molecular docking.The main results were as following.The nucleotide sequences of VL and VH were obtained from hybridoma cells(F12B8)which secrete monoclonal antibody recognizing ABA and conjugated ABA.And then the genes of VL and VH were constructed to scFv S12B8.The results of ELISA showed dose-dependent inhibition of the purified S12B8 protein by ABA,ABA-ME and ABA glucose ester tetra acetyl(ABAGE tetra acetyl).The detection limit(10%inhibition)of ABA,ABA-ME and ABAGE tetra acetyl was 19.7,8.1 and 4.0 ?g/ml,respectively.The inhibition rate of ABA-ME and ABAGE tetra acetyl was approximately 1.7 and 2.4 folds stronger than ABA.We investigated the interactions between S12B8 and ligands by homology modeling and molecular docking.The binding energy of ABA,ABA-ME and ABAGE to the S12B8 antibody was-37.1,-69.6 and-112.0 kcal/mol,respectively.The binding energy rate of ABA-ME/ABA and ABAGE/ABA to S12B8 was 1.87 and 3.0,which agreed well with our competitive ELISA results.