Proteomic And Physiological Phenotype Study On The Inhibiting Expression Of Ataxin-3 In N. Benthamiana

The transcript subtraction cDNA library was constructed with drought-treated potato leafs in our previous study.StATX-3,encoding potato ataxin-3 protein was identified by screening in the library and the full-length cDNA of StATX-3 was cloned.Through BLAST it was found that StATX-3 is located on chromosome 10 in potato genome.StATX-3 encoding protein(DMP400033234)was annotated as ataxin-3.The function of plant ataxin-3 has not been reported yet.In this study,transgenic lines,in which ataxin-3 expression is inhibited,will be obtained through transformation of antisense StATX-3 cDNA in N.benthamiana genome.Through assay and analysis on physiologic phenotype and proteomic profile of the transgenic lines,pathways StATX-3 involves in will be revealed.(1)With the vectors of pC-35S and pG-StATX-3,a recombinant vector pC-35S-StATX-3,aiming to inhibit expression of StATX-3 in vivo was constructed by subcloning.Through Agrobacterium-mediaed transformation,antisense StATX-3 cDNA was transformed into N.benthamiana genome.The transgenic line stax-1 and stax-7,in which ataxin-3 expression was significantly decreased were selected by the test on ataxin-3 expression(p<0.01).(2)Assay on the physiological phenotypes showed that the ATP content in stax-1 and stax-7 was significantly higher than that of the control wild type(WT),indicated that down-regulation of ataxin-3 expression increased ATP accumulation in N.benthamiana.At the same time,the chlorophyll contents in stax-1 and stax-7 leaves were significantly higher than that of the control(p<0.01),suggesting that the chlorophyll accumulation of leaves was increased along less expression of ataxin-3 in N.benthamiana.(3)Through statistic test and setting on threshold value,168 DAPs(differential abundance protein)from iTRAQ date,in response to ataxin-3 expression down-regulated were identified.Through GO functional classification and KEGG pathway enrichment analysis,it was determined that these DAPs were mainly involved in photophosphorylation and terpenoid metabolism.Futheremore,it was found that the accumulation of photophosphorylated ATP synthase such as ATPF and ATPE increases significantly in ataxin-3 inhibited lines,which was consistent with the results of physiological phenotype assay(p<0.01).In addition,the increase of terpenoid skeleton accumulation of chlorophyll synthesis substrate is consistent with the results of physiological phenotype assay on chlorophyll content.These results indicated that down-regulation of ataxin-3 expression enhanced the accumulation of photophosphorylated ATP as well as chlorophyll and other terpenoids in N.benthamiana.According to the previous reports that plant energy charge regulates drought tolerance,it is speculated that ataxin-3 involves in response to water deficit stress through regulation of energy charge level.