番茄果形调控基因fs8.1的图位克隆与功能分析

fs8.1 is an important fruit shape locus which does not only increase fruit shape index, but also increases fruit weight and pericarp thickness. Based on its advantages in the regulation of fruit shape and weight, fs8.1 has a high-potential value in the breeding of processing tomato. However, the fs8.1 gene has not been cloned so far, which is a great restriction on the illumination of its function, as well as the application of fs8.1 in processing tomato breeding. Therefore, in this project, by performing several experiments, we attempt to clone the fs8.1 gene and clarify its function. First, through marker development, fine-mapping, gene expression analysis and genome sequence variation analysis, we attempt to narrow down the introgression region of fs8.1 and select the candidate genes of fs8.1 within the region. Second, through gene overexpression, gene knockout, gene silencing and functional complementation, we attempt to clone the fs8.1 gene and illuminate its function at both transcriptional and histological level. Third, through fs8.1 specific marker development, allele distribution analysis and population structure analysis, we attempt to clarify the distribution of fs8.1 alleles in different tomato varieties. Last, through the development and analysis of fs8.1+sun+ovate near-isogenic lines (NILs) in Rio Grande background, we attempt to clarify the interaction of fs8.1 with other fruit shape elongation genes. The results obtained from this project are not only important for clarifying the mechanism of the fruit shape regulation and the plant organ morphogenesis, but also valuable for accelerating the tomato breeding as well as improving the tomato industry.

fs8.1是番茄中一个重要的果形调控位点，不仅可以均匀伸长果形，还可增加果重与果皮厚度，在加工番茄育种中有潜在的应用价值。至今fs8.1基因尚未被克隆，这一问题不仅严重阻碍了我们对其功能和调控机理的阐明，同时也极大的限制了其在育种中的应用。因此，本项目拟通过分子标记开发、基因精细定位、基因表达与序列变异分析，缩小fs8.1的定位区间并筛选出候选基因；之后通过稳定遗传转化进行基因过表达、沉默、敲除和功能互补试验，克隆fs8.1基因并明确其功能；而后通过特异性分子标记开发、等位基因与群体结构分析，明确fs8.1等位基因在不同类型番茄中的分布；最后，通过构建和研究以加工番茄为背景的fs8.1、sun和ovate三重近等基因系，阐明fs8.1与其他果形伸长基因的互作。本项目研究结果不但对阐明番茄果形调控机理和完善植物器官形态建成理论有重要的科学意义，而且也将对番茄育种和番茄产业有积极的推动作用。

我国是番茄生产与出口大国，其中加工型番茄在番茄产业占有重要地位。fs8.1是调控加工型番茄果形的重要位点，本项目对这一位点开展研究，以期克隆该基因并明确其功能以及与其他果形位点的互作。通过精细定位将fs8.1位点定位在8号染色体长臂末端270 Kb区域，其中含有三个注释基因。而后通过基因敲除验证Solyc08g061910为FS8.1基因，其编码一个Trihelix GTL1-like转录因子。而fs8.1突变是由一个SNP所引起的提前终止子所导致的。根据该突变开发了分子标记并在各种番茄材料中进行检测，发现fs8.1主要存在于加工番茄果形指数大于1的材料中，而野生与樱桃番茄中没有检测到该突变。fs8.1突变主要通过增加纵向细胞数来调控果形与果实大小。同时其也调控花瓣和萼片的形状、小叶数、叶片夹角、叶柄及茎髓部横切面的细胞大小。在品质方面，fs8.1还提高了果实的黏度和耐压力，增加了果实中的奎宁酸，降低了苹果酸含量。通过花蕾与子房发育研究，该基因在花形成早期起作用。通过转录分析可知fs8.1突变对花蕾的调控作用在16 dpi开始明显。fs8.1、sun和ovate在调控番茄种子大小中存在互作。单独的果形基因对种子大小影响不显著，但果形基因之间的互作会减小种子面积并影响种子形状指数，其中，sun/fs8.1面积减小是因为种子长宽同时减小，对种子形状指数没有影响；sun/ovate、ovate/fs8.1、sun/ovate/fs8.1面积减小是因为种子长度减小，宽度不变造成的，因此形状指数也减小。组织学分析发现，含有s/f、s/o和s/o/f位点的种子，胚所占比例小于野生型种子，而空腔所占比例和种皮所占比例差别不大。转录水平分析发现了一些受果形基因调控并可影响种子大小的模块，这些模块中的基因可能是番茄中3个果形位点的下游效应基因。本项目结果对阐明番茄果形调控机理、提高加工型番茄育种效率有积极的推动作用。