玉米自主性Helitron转座子的分子克隆与遗传特性研究

Genome sequencing projects have revealed that a large fraction of eukaryotic DNA consists of transposons. The computational analysis of genome sequences has led to the discovery of several novel types of transposons, yet only in a few cases have predictions regarding the biological activity of computationally identified transposons been supported by experimental data. Helitrons are rare transposons discovered by computational analysis, rather than genetic studies. Although they comprise an ancient superfamily of transposons found in eukaryotes, it is in plants where they have been studied extensively and identified as quite few putative autonomous elements and abundant nonautonomous elements with and without gene fragments. . The molecular structure of the autonomous Helitron and the postulated rolling circle mode of transposition have been unraveled experimentally in animal, and recent evidence suggests that Helitrons may transpose by cut-and-paste mechanism, at least in plant, without knowing the existence of the autonomous Helitrons and their molecular structures. Two Helitron properties, in particular, have caught the imagination of biologists: their ability to undergo sudden bursts of transposition and their ability to capture fragments from different genes to make chimeric transcripts. . Maize, the organism where transposons were discovered, continues to be an excellent model system for their study and a rich source of genetically defined, biologically active, transposon families that await molecular characterization. .Helitron transposons comprise around 1/16 of the maize genome, and generate tandem concatemers that amplified by RCR and accumulate mostly in centromeres. However, majority of Helitrons occur as solo elements in the genome. We have reported that Helitrons in maize may transpose by an alternative cut-and-paste mechanism, at least somatically.. The aim of this project is to take advantage of the broad variability from diverse maize genetic resource to isolate the mysterious autonomous Helitron transposon in plant. We have constructed a reporter of Helitron transposition activity in maize, in which the C1 gene is interrupted by a GFP labeled non-autonomous Helitron. When transformed into colorless aleurone lines or crossed with other colorless aleurone lines, this gene produces spotted kernels indicating the existence of autonomous elements and the cut-and-paste transposition mechanism. This information will, in turn, help on the molecular cloning of the autonomous element in the segregating population, contribute to our understanding of what the exact molecular structure of an autonomous element is in maize, how Helitron transposons transpose either by rolling-circle-replication or cut-and-paste mechanisms, and what the repairing mechanism and insertion preference of this novel DNA transposon.

Helitron是通过计算分析新发现的真核生物DNA转座子。6%的玉米基因组是Helitron，包括大量非自主性和少数潜在的自主性Helitron。序列分析推论Helitron以滚环复制扩增(rolling-circle-replication)的方式发生转座，但玉米中发现了以剪切粘贴(cut-and-paste)模式发生转座的Helitron。近来对人工构建的蝙蝠自主性Helitron进行了初步研究，植物的自主性Helitron却仍有待发现。本项目通过构建玉米C1基因插入GFP标记的非自主性Helitron的转座活性检测系统来分离鉴定玉米的自主性Helitron，在转化无色糊粉层玉米品系后，后代籽粒的着色差异将反应自主性Helitron的有无。玉米自主性Helitron的克隆对于揭示其详尽的分子结构、遗传转座机制发生的机理、插入序列的偏好性、基因进化及功能演变均具有重要意义。

Helitron最初是通过计算分析基因组文库中已有序列而发现的DNA转座子新类型。 Helitron是玉米等作物基因组中很重要的序列组成部分，在基因的调控和优异等位基因变异方面发挥着重要作用，但是，该类转座子的转座机制及其自主性转座子的序列特征和遗传调控机理在植物中均属未知领域。.本项目的主要研究内容包括：1、构建7个玉米Helitron转座子转座活性检测报告系统，即c1-dHel-GFP载体，其中玉米籽粒糊粉层细胞花青素合成途径C1基因插入了带有GFP标记的非自主性Helitron（defective Helitron, dHel），包括了启动子区域和编码区域等四个插入位点及四个不同家族的dHel。2、遗传转化后的转基因株系与数十个广泛遗传背景的玉米自交系杂交来鉴定并克隆玉米自主性Helitron（Autonomous Helitron, aHel），进而揭示植物基因组中此类崭新DNA转座子的序列组成、滚环复制和剪切粘贴转座机制、转座后修复机制和新插入位点的序列偏好性等遗传特征。.研究发现：1、不同构建载体的转基因表达因dHel插入位点和dHel的类型的不同而有所差异。只有插入在编码区起始密码子的dHel插入阻断了c1基因的正常表达谱，转基因籽粒为无色，其他载体中启动子区的dHel插入未引起C1基因丧失功能，同时产生了极为丰富的c1基因转录本（这是之前研究中未曾检测和发现的方面）包括c1基因的正常转录本，因此后代籽粒中有紫色表型。2、通过分子生物学手段，在部分杂交F1代（T2 × c1测试系）的幼叶中可以检测到dHel-GFP发生剪切转座后的精确修复产物，预示着c1测试系中可能存在某些类型的自主性aHel，同时表明，遗传转化后的dHel与内源dHel相似也可以通过“剪切粘贴”模式转座。3、在dHel精确剪切转座转基因遗传材料中检测到环状dHel-GFP序列的缺失变异类型，表明dHel-GFP在体细胞中还可能存在滚环复制的转座模式。.因此，本项目的Helitron转座子转座活性检测系统在分子水平上展示了玉米中aHel的遗传活性及其在体细胞中的多种转座模式，首次揭示了Helitron插入引起宿主基因转录水平的诸多变异，为最终克隆自主性Helitron奠定了坚实的遗传材料基础和实验依据，探索出了切实可行的实验思路与技术手段。