Functional Identification Of NtabSPL6-1 In Nicotiana Tabacum And Preliminary Screening Of Its Interacting Proteins

The main contents of this research included functional analyses of NtabSPL6-1 and NtabSPL6-4,two genes isolated from Nicotiana tabacum L.cv.Qinyan95,and screening of the interacting proteins of NtabSPL6-1.First of all,transgenic Arabidopsis and tobacco plants constitutively expressing NtabSPL6-1 and NtabSPL6-4 were acquired by genetic transformation mediated by Agrobacterium tumefaciens,and the phenotypes of NtabSPL6-1 transgenic plants have been observed and analyzed.On this basis,proteins which possibly could interact with NtabSPL6-1 were preliminarily identified through CoIP-MS method,and a bioinformatic analysis has been made as well.Before the genetic transformation experiments,the coding sequences of NtabSPL6-1 and NtabSPL6-4 were amplified with the designed specific primers,and the overexpression vectors and the GFP subcellular localization vectors of these two genes were constructed.Then the recombinant plasmids were introduced into Agrobacterium strains GV3101 and LBA4404,and wild-type Col-0 Arabidopsis plants and Qinyan 95 tobacco leaves were transformed using floral dip and leaf disc methods,respectively,to obtain the transgenic plants.By phenotypic observation,it was found that the constitutive expression of NtabSPL6-1 in Arabidopsis could promote the development of trichomes in leaf epidermis and influence the growth patterns of leaf.In tobacco,overexpression of NtabSPL6-1 led to dwarfism of the plants,and could change the morphological structure of leaves and the development of glandular hairs in leaves.Based on the previous reports,it could be speculated that NtabSPL6-1 could affect the the resistance of plants to stress factors indirectly by regulation of the growth process.In the present work,the polyclonal antibody of NtabSPL6-1 was prepared and was used in preliminary screening of the interacting proteins of NtabSPL6-1 by CoIP-MS techniques.By bioinformatics methods,these proteins were categorized and annotated from three aspects,including biological process(BP),cell component(CC)and molecular function(MF).The results showed that these proteins were mainly involved in several biological processes,including cellular process,cell metabolism process and cell macromolecular metabolism process,and were basically localized within the cell.It is important that some proteins participate in plant-pathogen interaction pathway,phytohormone signal transduction pathway,and glucosinolate biosynthetic pathway.In combination with previous reports and the phenotypic observation of the transgenic plants,it could be speculated that these proteins may have interaction with NtabSPL6-1.To lay a foundation for further screening of the interacting proteins by yeast two-hybrid system,a cDNA library of tobacco leaf was established in the present work.At the same time,bait vectors of NtabSPL6-1 were constructed and bait peptide fragments were determined by testing of the autoactivation capability.The constructed bait vectors carry the intact coding sequence of NtabSPL6-1,the coding sequence of SBP-C terminal domain,the coding sequence of C terminal domain,the coding sequence of N terminal domain and the coding sequences of three different regions in N terminal domain.The results indicated that SBP-C terminal domain and C terminal domain of NtabSPL6-1 possessed strong transcriptional activation ability,the intact protein,N terminal domain and N1 peptide fragment possessed weak transcriptional activation ability,N2 and N3 peptide fragments had no transcriptional activation ability.On account of the weak transcriptional activation ability showed by the intact NtabSPL6-1 and the strong transcriptional activation ability showed by SBP-C terminal domain and C terminal domain,it could be concluded that NtabSPL6-1 possessed self-regulation capability and its N terminal domain could block the activity of its C terminal domain.Tobacco NtabSPL6-1 might play a role in the signal transduction pathways mediated by plant hormones.It could affect the development process of plant trichomes and possibly played important functions in tobacco resistance to insect pests.It is reasonable to believe that there are a number of proteins interacting with NtabSPL6-1 in tobacco,and they might participate jointly in regulation of the defense pathways of tobacco against herbivores and microbial pathogens.However,the details of these interacting proteins and the defense mechanism remain to be further explored.