白桦BpGT14基因启动子克隆及表达活性分析

发布时间：2018-09-08 10:43

【摘要】：本文利用Site Finding-PCR方法克隆了白桦BpGT14基因起始密码子ATG上游2 169 bp序列,并通过PLACE启动子预测工具对其进行元件分析。结果表明,该启动子片段含有启动子核心元件及多种逆境及激素响应元件,同时具有植物苯丙烷及木质素生物合成的MYB类转录因子的重要结合基序。研究选取了其中含有启动子核心元件的1 156 bp片段构建了pBpGT14∷GUS植物表达载体,利用农杆菌侵染的方法将pBpGT14∷GUS报告基因瞬时转化烟草植株,鉴定该启动子在烟草中的表达活性及对非生物胁迫和激素的响应模式。对转基因烟草植株进行GUS染色,结果表明该启动子具有启动活性,且在茎段处活性较高;进一步分析非生物胁迫对烟草中GUS酶活性的影响,表明该启动子对ABA、NaCl、PEG及高温处理均有明显响应,且对于NaCl及PEG处理响应迅速。为了更好的鉴定白桦BpGT14基因启动子在白桦细胞中的启动活性及响应模式,本文构建了pBpGT14∷GFP载体并瞬时转化白桦茎段悬浮细胞,进行研究。GFP转录水平分析结果与GUS酶活性结果基本一致,但其中部分时间点仍存在差异。选取PEG处理3、6、12及24 h的转GFP基因白桦茎段悬浮细胞,在显微镜下观察其绿色荧光蛋白,以此揭示该启动子对干旱的响应模式。结果表明,该启动子在白桦茎段悬浮细胞中启动了GFP的表达,在处理初期(3 h),荧光效果明显;随着处理时间的增加,细胞脱水明显,且在细胞壁表现高亮度荧光。
[Abstract]:In this paper, the upstream 2 169 bp sequence of Bai Hua BpGT14 gene initiation codon (ATG) was cloned by Site Finding-PCR method and analyzed by PLACE promoter prediction tool. The results showed that the promoter fragment contained promoter core elements, various stress and hormone response elements, as well as important binding motifs of MYB transcription factors synthesized by plant phenylpropane and lignin biosynthesis. PBpGT14: GUS plant expression vector was constructed with 1 156 bp fragment containing promoter core element. The pBpGT14: GUS reporter gene was transiently transformed into tobacco plant by Agrobacterium tumefaciens infection. The expression activity of the promoter in tobacco and its response to abiotic stress and hormone were identified. The results of GUS staining on transgenic tobacco plants showed that the promoter had high activity at stem segments, and the effects of abiotic stress on the activity of GUS in tobacco were further analyzed. The results showed that the promoter had obvious response to ABA,NaCl,PEG and high temperature treatment, and rapid response to NaCl and PEG treatment. In order to better identify the promoter activity and response pattern of Bai Hua BpGT14 gene promoter in Bai Hua cells, pBpGT14: GFP vector was constructed and the suspension cells were transformed into the suspensions of Bai Hua stem segment. The results of transcriptional level analysis of .GFP were consistent with the results of GUS activity, but there were still some differences at some time points. In order to reveal the response pattern of the promoter to drought, the suspension cells of GFP gene Bai Hua were treated with PEG for 12 and 24 hours, and the green fluorescent protein was observed under microscope. The results showed that the promoter initiated the expression of GFP in the suspension cells of Bai Hua stem segment, and the effect of 3 h), fluorescence was obvious at the initial stage of treatment, and with the increase of treatment time, the cells were dehydrated obviously and showed high brightness fluorescence in the cell wall.
【作者单位】：东北林业大学生命科学学院林木遗传育种国家重点实验室;
【基金】：中央高校基本科研业务费专项(2572014DA04) 国家自然科学基金项目(31200463,J1210053)
【分类号】：S792.153

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