胁迫诱导苹果内源逆转座子atr1活性表达的甲基化调控机制及其转座标签研究

For promoting the development of apple functional genomics and bud mutation breeding, the present study is designed to reveal the the role of cytosine methylation of atr1 (an isolated transposable endogenous retrotransposon in apple genome) DNA on transposition activity and to establish the methodologies of the retrotransposon atr1 tagging. The retrotransposon atr1 was inactive under normal conditions and was activated under stresses. Based on the stresses transcriptionally activating atr1, stress situations were used to activate the retrotransposon atr1 in 'Delicious' apple. The atr1 DNA methylation sites, degree, and status and the atr1 transcriptional activation were detected by bisulfite sequencing PCR (BSP), Southern-blot, and qRT-PCR, respectively, for investigating the relationships between atr1 transcriptional activity and its methylation states during the treatments of stress conditions. The samples of methylation alteration of the atr1 DNA were used to detect the atr1's transpositional insertion sites in mutants for clarifying the possible roles of cytosine methylational on atr1's activity, and obtaining the conditions controlling the transpositional insertion frequency of the atr1 in mutants and the atr1 transpositional insertion mutant lines. The mutants were used to establish the system amplifying the insertion sites and the sequences flanking the insertion site. The mutants and the 'Delicious' clone were used to isolate the insertion mutated-genes. The isolated genes were submitted to GenBank for predicting the function. The screening and characterization of the mutants in the field were combined with the predicted-genes function for uncovering the biological functions of the mutated genes discovered by the atr1 tagging.

为促进苹果功能基因组学研究，开展基于逆转座子的芽变育种工作，揭示具有转座活性的苹果内源逆转座子atr1活性表达调控机理，创建突变体并建立atr1转座标签，拟以获得atr1的"元帅"为试材，借助已证实的atr1转录激活胁迫条件，分别利用BSP、Southern杂交和qRT-PCR检测胁迫处理过程中atr1甲基化位点、水平、模式与状态的变化、转座插入位点的变化以及表达活性的变化，分析不同胁迫作用的甲基化位点与胁迫过程中atr1甲基化变化规律；解析atr1甲基化与表达活性相关性，阐明胁迫激活atr1的甲基化调控机制；获得调控atr1转座插入频率的胁迫条件及atr1转座插入突变体；建立atr1转座标签体系分离突变体中转座插入位点侧翼序列，分离突变基因；结合突变体田间表现分析变异性状，预测突变基因功能。本研究将为利用逆转座子atr1构建苹果突变体库，培育芽变品种及开展苹果功能基因组学研究奠定基础。

前期研究结果显示逆转座子参与了元帅系品种的短枝变异，本项目利用基于逆转座子的IRAP和染色体步移技术揭示导致元帅系短枝变异的是一长度为2190 bp的逆转座子Mdsolo-LTR1插入所致，插入位点侧翼存在多个功能基因；苹果基因组中存在相似度高的多拷贝Mdsolo-LTR1及其同原序列； Mdsolo-LTR1具有双向启动子活性，且茎中的表达活性高于根中；其正、反义链不同长度的缺失突变体均具有启动子功能，初步鉴定出Mdsolo-LTR1正、反义链中增强和抑制活性表达元件所在区域，已获得Mdsolo-LTR1正、反义链不同长度的缺失突变体转化的拟南芥T2或T3代株系，将用于Mdsolo-LTR1正、反义链中启动活性和表达活性调控元件的鉴定及其对不同胁迫响应的分析，这将有助于解析Mdsolo-LTR1及其同原序列对苹果基因组进化的作用及苹果基因组如何利用这些元件促进自身进化。已获得短枝突变体中Mdsolo-LTR1插入位点侧翼序列中功能基因的异源转化植株，这为阐明苹果基因组中哪些基因在Mdsolo-LTR1的影响下参与了苹果的短枝突变提供了试材。逆转座子atr1（Mdoryco1-1）在ABA、2,4-D、伤害（Wn）处理后表达活性增强，甲基化分析结果显示多数胁迫处理后其5’-LTR正义链中CHG和CHH位点甲基化水平增加；伤害、2,4-D和ABA处理后mCG甲基化水平没有变化，但SA处理后下降。由于苹果基因组内含有多个拷贝的Mdoryco1-1及其LTR同源序列，增加了胁迫处理条件下甲基化分析的难度，已将Mdoryco1-1 LTR正反义链分别转至烟草中以便于进一步分析。苹果基因组内Mdoryco1-1拷贝数低且临近序列多为功能基因区。异源瞬时表达分析显示Mdoryco1-1的5’-LTR和3’-LTR都具有双向启动子功能。