Functional Analysis Of A Nicotiana Benthamiana Ubiquitin Extension Protein Gene

Ubiquitin is a ubiquitously existed small molecular protein of 76 amino acids.It degrades proteins with ubiquitylation modification in 26S proteasome-dependent way, or alters protein localization,activity and interactions through interacting with proteins with ubiquitin binding domain in 26S proteasome-independent way,thereby regulates a variety of biological processes.Ubiquitin extension protein(UEP)is a fusion protein of an ubiquitin molecule in the amino terminal and a ribosomal protein molecule in the carboxyl terminal.The regulatory role and its mechanism of UEP in plant biological processes are still unclear.In this thesis study,an UEP gene was cloned from Nicotiana benthamiana.Its role in development,necrosis,disease resistance and stress tolerance in N.benthamiana was studied employing virus induced gene silencing(VIGS)and overexpression techniques.The main results are as follows:1.An UEP gene was cloned from Nicotiana benthamiana and designated as NbUEP.It had an open reading frame of 471 bp.NbUEP was a fusion protein of an ubiquitin molecule of 76 amino acids in its amino terminal and a s27A 40S-type ribosomal protein molecule of 80 amino acids in its carboxyl terminal.The nucleotide sequence of NbUEP had the highest identity,96%and 91%respectively,to an N. tabacum UEP gene TUQG3(AJ223329)and an N.benthamiana UEP gene (AY912494),with difference of only 15 and 42 nucleotides,respectively.2.Effect of treatments with plant disease resistance elicitors and inoculations with pathogens on NbUEP gene expression was investigated.Expression of NbUEP and its components NbUBI and NbRLP in N.benthamiana plants was altered upon spraying treatments with solutions of plant disease resistance elicitors BTH,SA and ethyphon and inoculations with a fungal pathogen Sclerotinia sclerotiorum and an oomycete pathogen Phytophthora parasitica var.nicotianae.The alteration of expression patterns of NbUEP and its component NbUBI by the treatments and inoculation was very similar. 3.Functions of NbUEP in development,necrosis and disease resistance in N. benthamiana were analyzed employing Tobacco rattle virus(TRV)induced gene silencing technique.TRV-induced VIGS induction treatments for NbUEP and its component NbUBI led to rapid necrosis,resulting in whole plant death 7-8 days post agro-infiltration;while the treatment for Nb UBI did not cause systemic necrosis,but severely inhibited plant growth and development,making the newly formed leaves deformed and unable to develop into full leaves,and completely blocked Avr4/Cf-4-dependent hypersensitive response.4.The role of NbUEP in necrosis,disease resistance and salt tolerance in N. benthamiana were examined employing overexpression technique.Transient overexpression of NbUEP caused strong necrosis,while transient overexpression of NbUBI and NbRLP did not result in any necrosis.Overexpression of all three genes enhanced disease resistance of the oomycete pathogen Phytophthora parasitica var. nicotianae but not to the fungal pathogen Sclerotinia sclerotiorum.Aditionally,high concentration of NaCl compromised the NbUEP-mediated necrosis.Overexpression of all three genes decreased salt tolerance in N.benthamiana.Taken together,NbUEP may play an important role in development,necrosis, disease resistance and stress tolerance in N.benthamiana through functioning of its components NbUBI and NbRLP in both separate and interactive ways.