应用酵母单杂交技术筛选拟南芥CCT1互作转录因子研究

来源 :安徽农业大学 | 被引量 : 0次 | 上传用户:xiaochouya87
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Plants are sessile organism and are constantly affected by pathogens.Therefore,plants have evolved complex mechanisms to protect themselves from pathogen invasion.Transcription factors(TFs)play important roles in regulating their upstream target signalling pathways in plant defense.B-box(BBX)is a member of zinc finger proteins with one or two B-box,CCT or VP domains.The CCT domain-containing family of the BBX TFs have been implicated to be involved in several vital roles in regulating seed germination,flowering,biotic or abiotic stress response,plant hormone signal transduction among several other life activities.Bioinformatics methods were used to identify a CCT domain-containing(CCT1)in Arabidopsis thaliana genome.CCT1 cis-elements,domain analysis,evolutionary relationships,and gene expression patterns were analysed.Previous studies on BBX TF family have shown that these TF family are involved in biotic and abiotic stress.However,no studies have been made to discover other TFs that regulates CCT domain-containing genes.In this study,we describe a yeast one-hybrid library to screen transcription factors that regulate CCT1 TF expression in Arabidopsis thaliana based on a high quality yeast hybrid c DNA library construction and interaction analysis.The main results are as follows:1.Cloning and bioinformatics analysis of CCT1 promoter Primers were designed to clone the promoter sequence of CCT1 gene,the full-length of promoter sequence of CCT1 gene is 2052 bp.Domain analysis showed that it contains a CCT domain.Cis-element related to hormonal,biotic and biotic stress such ABRE,CGTCA-motif,DRE and W-box were identified in the CCT1 promoter sequence.CCT1 gene promoter was cloned into the p Ab Ai vector by recombinant reaction.2.Three transcription factors were identified to be the candidate regulators of the CCT1gene.Y1 H c DNA library constructed using the high-quality RNA from Arabidopsis was used for the Y1 H screening.15 transcription factors were initially identified and later were rescreened by Y1 H.The transcription factors including NAC53,CBF2 and CBF3 were found to directly bind to the CCT1 promoter.3.The three regulatory genes binds to different region in the CCT1 promoter.CBF2 and CBF3 TF binds to a motif located in the-1218 bp ~-270 bp upstream from the ATG starting point of the CCT promoter sequence.In addition,the CBF3 TF also binds to a motif located in the-1218 ~ 2117-bp upstream from the ATG starting point of the CCT promoter sequence whereas NAC53 TF binds to-1~-165 bp and-165 ~-270 bp upstream from the ATG starting point of the CCT promoter sequence.4.CCT1 and three candidate regulators had differential gene expression uponexogenous hormone treatment Real-time quantitative PCR technology to analysis the expression of CCT1,NAC53,CBF2 and CBF3 genes were analysed upon salicylic acid(SA),abscisic acid(ABA)and methyl jasmonate(Me JA).Upon SA treatment,the relative expression of CCT1,NAC53,CBF2,and CBF3 was upregulated.The relative expression upon ABA treatment was upregulated in CCT1,NAC53,CBF3 but not CBF2.A similar expression was observed with Me JA treatment.In summary,we identified three transcription factors that bind to CCT1 promoter using Y1 H technique.These three TFs binds to different motif in the CCT1 promoter.The three TFs showed diverse expression upon exogenous hormone treatment.This research provides new insights to understand the regulation of CCT1 gene under different hormonal stress.The interaction between these candidate TFs and CCT1 to enhance plant disease resistance needs further research.
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