Identification Of Potential Cargo Proteins Of The Arabidopsis Thaliana AtTRN1 And More Branch Phenotype Analysis Of AtTRN1 Konck-down Mutant Sic1

Nucleo-cytoplasmic trafficking is essential for eukaryotic cell functions survival.Transportin 1(TRN1),as a special importin? member,recognizes a special nucleus localization signal,so called PY-NLS and mediates nuclear import of a subset of cargo proteins in mammalian and yeast cells.Recent findings suggest that the cargoes of human TRN1(Kap?2),are involved in multiple aspects of cellular processes,including alternative pre-mRNA splicing,RNA transport,regulation of transcription,and protein translation.However,very few cargo proteins and functions of plant TRN1 have been identified until now.1.In this thesis,we first screened the cargoes poteins of AtTRNl,and obtained the following results:1).Firstly,we screened for proteins that interacted with AtTRN1 using Y2 H approach,and 18 candidates of AtTRN1 cargo proteins were identified.Sequence analysis indicated that no typical PY-NLSs motif was present in these 18 proteins.2).Further screening for the homologs of cargo proteins of yeast Kapl04 p and mammalian Kap?2 in Arabidopsis proteome were performed sequentially.Seven additional potential cargo proteins were identified.3).Then screening for the PY-NLS motif-containing proteins in Arabidopsis proteome were performed sequentially.four additional potential cargo proteins were identified using this strategie.4).The interaction between all the 29 potential cargo proteins and AtTRN1 were confirmed by both Y2 H and bimolecular fluorescence complementation(BiFC)assays.We next got another result that fragment include the PY-NLS motifs that will interacted with At TRN1.In previous screening experiment,we identified a AtTRN1 konck-down mutant sic1 in Arabidopsis thaliana,which exhibited multiple deficiencies and abnormalities In this study,we analyzed why sic1 have more branch than WT through physiological,and biochenmical and molecular techniques.The main results are as follows.1.sic1 plants have more branches than wild type plants,including rosette branches cauline branchs.The histological section and scanning electron microscope results suggested that the overall structure of SAM(shoot apical meristem)was as same in WT and sic1 mutant plants.In addition,sic1 plants and WT plants had approximately equal numbers of rosette nodes.These results demonstrate that the dormant axillary buds in sic1 plants were activated,and subsequently result in the increase of the branches.2.Apply of exogenous auxin IAA and NAA to sic1 plants,or overexpression of the endogenous auxin biosynthesis gene YUCCA4 in sic1 plants can effectively reduce the branches.These findings indicated that auxin can inhibit branching in sic1 plants,and maintain the axillary buds in dormancy.3.Exogenous cytokinin KT can increase the cauline branches of sic1 plants,whereas inhibitors of cytokinin LVST can reduced the cauline braches.Impacts to rosette branches were not observed with cytokinin or its inhibitors.These suggested that cauline branches were sensitive to cytokinin in sic1 plants.4.In grafting experiment,sic1 stock can restore the phenotype of strigolactone deficient mutant scion,and WT stock can not influence the sic1 scion.These results show that sic1 mutant can synthesize strigolactone.5.Apply of exogenous strigolactone GR24 to sic1 will reduce the number of rosette leaves,and this suggesting that the signal transduction pathway of strigolactone was not affected in sic1.6.In our microarray experiment,auxin-responsive genes were down-regulated at axillary bud,this phenomenon will be the result of decrease of auxin at these sites;Biosynthetic genes of strigolactone were also down-regulated,whereas signal transduction genes of strigolactone pathway were up-regulated,these results demonstrate that strigolactone contents may be changed at axillary bud in sic1 plants.The expression levels of cytokinin-biosynthesis genes and cytokinin-activated genes were decreased,the expression levels of cytokinin-metabolism genes and cytokinin-inactivated genes were increased.This result suggested that the content of cytokinin may be lower at axillae sites in sic1.Most of the ribosome-related genes were activated,demonstrating that ribosome assembly in these regions may be more active.In conclusion,AtTRN1 regulate various developmental processes of plants,especially for plant morphogenesis,through mediating nuclear import of diversified proteins.This study has not only the important scientific,but aslo provides a certain basis for research nucleo-cytoplasmic trafficking in plant.