Cloning And Functional Study Of NfD3,the Key Gene In The Signal Transduction Of Strigolactones From Nervilia Fordii

ObjectiveStrigolactones are a class of plant hormones that can regulate tillering,and MAX2/D3 is one of the key proteins involved in its signaling pathway.Based on the research progress of Arabidopsis thaliana,rice,wheat and petunia,NfD3,the homologous gene of MAX2/D3 in strigolactones signal transduction pathway,was cloned from endangered medicinal plant Nervilia fordii.The molecular regulation mechanism of NfD3 in strigolactones signal transduction was studied,which helps laying the foundation for finding approaches to reduce the resources shortage of N.fordii.MethodsThe NfD3 and NSK genes were screened in the transcriptome of N.fordii and cloned from the plant by the RT-PCR technology.NfD3 is highly homologous with the Arabidopsis AtMAX2 and rice OsD3 genes while NSK genes are highly homologous with the Arabidopsis ASK1 gene.The yeast two-hybrid technology and bimolecular fluorescence complementation were used to investigate the interaction between NfD3 protein and NSK proteins.NfD3 gene was transformed into Arabidopsis max2 mutant by floral dipping to observe whether the branching,primary root length and plant height phenotypes could be restored.The responses of NfD3 gene under salt stress was further studied and the interaction between NfD3 protein and ASK1 protein of Arabidopsis was investigated by yeast two-hybrid technology,to find out whether the function of NfD3 gene was similar to that of Arabidopsis AtMAX2 gene.Results1 NfD3 was cloned from N.fordiiThe NfD3 gene was successfully cloned from N.fordi with an open reading frame of 2073 bp.NfD3 encodes a protein with 690 amino acids,which belongs to the F-box protein family,containing conserved F-box and LRRs domains.The NfD3 locates in the nucleus,which is an intranuclear protein.Phylogenetic tree showed that the MAX2/D3 proteins of Phalaenopsis equestris and Apostasia shenzhenica were the closest to NfD3.2 NfD3 interacts with NSK2,NSK3,NSK7 and NSK11 proteins of N.fordiiFive NSK genes were successfully cloned from N.fordii,namely NSK2,NSK3,NSK5,NSK7,and NSK11,respectively.NSK proteins all have F-box and Cullin protein binding sites and belong to the SKP1 protein family.NSK2,NSK3 and NSK5 located in the nucleus,while NSK7 and NSK11 located in the cytoplasm and nucleus.NSK2,NSK3,and NSK5 were clustered together,and they were closely related to SKP1 proteins of Triticum aestivum and Zea mays;while NSK7 and NSK11 clustered together and were both closer to SKP1 proteins of Phytophthora polycephalum.Results of the preliminary screening using yeast two-hybrid technology showed that,NSK2,NSK3,NSK7 and NSK11 proteins may interact with NfD3.The bimolecular fluorescence complementation analysis in tobacco cells further confirmed NfD3 protein may be involved in the formation of SCF complex by binding with NSK2,NSK3,NSK7 and NSK11 proteins.3 NfD3 regulates plant branching,root development and salt toleranceNfD3 gene was transfected into Arabidopsis max2 mutant by floral dipping.Ectopic expression of NfD3 in max2 mutant restored the primary rosette branches and primary root length deficiency phenotypes of max2 mutant to the wild type,indicating that NfD3 can compensate the function of AtMAX2 in regulating the branching and primary root length of plants.In addition,in comparison with the max2 mutant,35::EGFP-NfD3/max2 exhibited significant salt tolerance,reduced the inhibition effect of high salt stress on the primary root length of max2 mutant,showing that NfD3 gene can reduced the sensitivity of max2 mutant to salt stress,and play the pivotal role in the regulation of salt stress resistance.Moreover,yeast two-hybrid technology showed that the F-box region of NfD3 protein could interact with ASK1 protein of Arabidopsis,suggesting that NfD3 protein may participate in SLs signal transduction by binding with ASK1 protein to form SCF complex,and then regulate the branches,primary root development,and salt tolerance of Arabidopsis.ConclusionsThe key gene NfD3 of the SLs signal transduction pathway was cloned from N.fordii.This gene encodes a protein can bind with NSK2,NSK3,NSK7,and NSK11 proteins of N.fordii,which provides the evidence of its participation in the formation of SCF complex.Heterologous expression of NfD3 in Arabidopsis max2 mutant showed that NfD3 not only control shoot branching and primary root length but,most importantly,regulate salt adaptation,further confirming the biological functions of NfD3 as AtMAX2 homolog in Arabidopsis.