【作者】 刘羽佳；

【导师】 王玉成；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2013， 博士

【摘要】 bHLH (basic/helix-loop-helix)家族是植物转录因子中最大的家族之一,它们的结构特性是都含有一个由60-100个氨基酸构成的高度保守的DNA结合结构域,包含2个功能不同的区域,即basic区域和HLH区域。绝大多数植物bHLH转录因子能够通过识别G-box元件参与诸如生长发育、营养吸收、信号转导、抗逆境胁迫等方面的应答。本研究通过对AtbHLH112基因过表达、抑制表达、野生型以及AtbHLH112基因缺失突变体植株在ABA、NaCl和Manntiol三种胁迫下的比较分析,证明AtbHLH112过表达植株的抗逆能力明显提高,说明AtbHLH112基因在拟南芥植株内发挥着非常重要的抗逆功能。利用基因枪瞬时转化技术将AtbHLH112与GFP的融合表达载体pROK2-AtbHLH112-GFP转化入洋葱表皮进行亚细胞定位,结果表明AtbHLH112转录因子定位在细胞核中。将AtbHLH112基因上游1320bp的启动子与GUS融合构建植物报告表达载体p1301-AtbHLH112并转化拟南芥,GUS化学染色表明,AtbHLH112基因的启动子具有强的表达活性,且具有组织特异性。通过PLACE和PlantCARE数据库预测分析发现,AtbHLH112基因启动子序列上含有ARR1TA、GTGANTG10、 MYCCONSENSUSAT、MYBCORE、MYBST1和WRKY71OS等与信号传递途径相关以及植物抗逆相关的顺式作用元件。通过酵母单杂交分析筛选到能够特异识别AtbHLH112基因启动子上E-box元件和W-box元件,从而调控AtbHLH112表达的上游基因各1条,即AtPP2C24和AtWRKY66.进一步的荧光实时定量RT-PCR结果表明,AtPP2C24和AtWRKY66基因在胁迫下的表达模式与AtbHLH112基因相似,该结果也提示三个基因可能属于响应逆境胁迫的同一表达调控网络。利用酵母单杂交等技术对AtbHLH112与顺式作用元件G-box的互作研究结果表明,AtbHLH112转录因了能够特异性地识别G-box顺式作用元件,且AtbHLH112转录因子的DNA结合域靠近基因C端。利用Agilent拟南芥全基因组表达谱芯片技术对AtbHLH112基因过表达植株与突变体植株在正常和盐胁迫条件的差异基因进行比较分析。非胁迫条件下共得到差异表达基因4159条(P<0.05；FC>2)；盐胁迫下共得到差异表达基因2862条(P<0.05；FC>2)。结果显示：AtbHLH112基因能够调控一系列相关基因的表达,特别是核糖体蛋白、热激蛋白、转导素蛋白、细胞周期蛋、抗病性蛋白的相关基因以及多种转录因了,如NAC、 WRKY、DOF、MYB、ZFP、ERF、TCP等等。酵母双杂交分析结果显示,AtbHLH112转录因子单体具有转录自激活活性,且自激活结构域靠近基因N端。将不含转录自激活结构域的AtbHLH112片段与拟南芥cDNA文库菌株进酵母双杂交,共得到7个能够与AtbHLH112转录因子互作的蛋白,包括2个叶绿素结合蛋白、2个泛素结合酶、1个谷氨酰胺合成酶、1个脂氧合酶和一个未知蛋白。更多还原

【Abstract】 The basic helix-loop-helix (bHLH) genes are a big group of transcription factors in plants. which is made up by60-100amino acids, containing a highly conserved DNA binding domain is followed by basic region and HLH.11domain. In plants, the vast majority of bHLH genes involved in regulating response by identifying the cis-acting element of G-box, such as growing development, nutrient absorption, signal transduction and adversity stress. In the present study, the T3transgenic Arabidopsis overexpressing AtbHLH112showed considerable stress tolerance under abiotic stress of ABA, NaCl and Manntiol, by comparing with T3transgenic Arabidopsis of AtbHLH112suppress expression, T3deletion mutant Arabidopsis of AtbHLH112, and wild type of Col-0. The recombinant AtbHLH112-GFP fused gene was transformed into onion epidermal cells by particle gun for subcellular location analysis. The results showed that AtbHLH112is located in nuclear of cell. The AtbHLH112promoter was fused with GUS. and introduced into wild-type Col-0by floral infiltration for promoter activity analysis. And GUS staining analysis indicated that the promoter of AtbHLH112has high activity in different plant tissues. PLACE and PlantCARE database prediction analysis suggested that there are many cis-elements which associated with signaling pathway and strss resistance of plants in the promoter of AtbHLH112, such as ARR1TA. GTGANTG10, MYCCONSENSUSAT. MYBCORE. MYBST1and WRKY71OS. We obtained that a AtPP2C gene (AtPP2C24) and a AtWRKY gene (AtWRKY66), using yeast one-hybrid analysis, can regulate the expression of AtbHLH112through recognizing E-box and W-box in the promoter of AtbHLH112, respectively. Furthermore, real-time quantification RT-PCR showed that AtPP2C24and AtWRKY66shared a similar expression patterns with AtbHLH112in response to adversity stresses, indicating that they may belong to the same expression regulation network. Yeast one-hybrid study showed that AtbHLH112can interact with G-box, and the DNA binding domain located in C-terminal of AtbHLH112. Agilent Arabidopsis oligo microarray analysis was employed to identify differences in gene expression between transgenic Arabidopsis and deletion mutant of AtbHLH112under normal and salt stress conditions. The results revealed that there are4159genes and2862genes significantly (P<0.05; FC>2) differentially expressed, respectively, under normal and salt stress conditions. The Agilent Arabidopsis oligo microarray results showed that AtbHLH112can regulate series related target genes, especially the related genes of ribosomal protein, heat shock protein, transduction grain protein, cell cycle protein, disease-resistance protein as well as a variety of transcription factors, such as NAC. WRKY. DOF, MYB, ZFP, ERF, TCP. and so on. Yest two-hybrid analysis revealed that the single form of AtbHLH112has transactivation activity, and the domain located in N-terminal of AtbHLH112; the fragment of AtbHLH112which do not contain transcriptional activation domain hybridized with the cDNA library of Arabidopsis thaliana to identified7interacting proteins, including2chlorophyll binding proteins,2ubiquitin-conjugating enzymes, a glutamine synthetase. a lipoxygenase and an unknown protein.更多还原