黄瓜小叶突变体Cssl基因的精细定位、克隆和利用

Leaf size is an important agronomic trait that directly affects crop yield, population microenvironment and resistance to water deficit. However, the excellent genes that under control of leaf size are very limited currently. Cultivated cucumber is very sensitive to water deficit due to the large leaves, and causing inconvenience in cultivation and management. Meanwhile the large leaves make the canopy structure unreasonable, the ventilation and transmissibility decreased and the diseases increased in the middle and late stage of the cultivated population, which directly affects the cucumber quality and yield. So appropriate reduction of cucumber leaf area and selection of small leaf varieties have a great significance in cucumber safe and efficient production, which bases on the exploitation and utilization of the small leaf Gene. Our project team found an excellent cucumber mutant which was indistinguishable from other agronomic traits of wild-type except the small leaves, and have located the candidate gene (tentatively named Cssl) in the region of 98kb without the known genes related leaf development or leaf size. The project intends to use fine mapping, qPCR, microscopic observation and genetic transformation techniques to validate and resolve function of candidate gene Cssl; and utilize the gene by transformation. The results not only help to reveal the molecular mechanism of cucumber leaf size development, but also accelerate the process of small leaf varieties breeding and provide a new theoretical basis and genetic resources for the molecular breeding of cucumber ideal plant type.

叶片大小是重要的农艺性状，直接影响作物产量、群体微环境及对水分胁迫的抗性等。但目前挖掘的控制叶大小的优异基因却非常有限。栽培黄瓜叶大，对水分亏缺极为敏感，同时大叶使栽培群体中后期冠层结构不合理，通风透光下降，病害发生增加，直接影响黄瓜品质和产量。选育小叶型的品种对黄瓜安全高效生产具有重要意义。挖掘小叶基因并对其利用是创制小叶品种的基础和前提。项目组从自交系中发现了叶小、抗旱性显著提高、其它农艺性状与野生型无显著差异的优异突变体，并将突变基因（暂命名Cssl）定位在118kb的区段，该区间不含目前已知的叶发育或叶大小相关基因。本项目拟采用精细定位、基因时空表达分析、显微组织结构分析和遗传转化等技术验证和解析Cssl候选基因的功能；同时借助于分子标记辅助选择，通过回交转育该基因到骨干亲本中。研究结果不仅有助于揭示黄瓜叶大小发育的分子机制，而且为黄瓜理想株型的分子育种提供新的理论依据和基因资源。

叶片大小是重要的农艺性状，直接影响黄瓜品质和产量。目前已克隆控制叶大小的基因甚少。本项目以一个心形小叶的黄瓜突变体为材料，开展候选基因精细定位、克隆和利用研究。突变体的心形叶是由于叶脉夹角小；叶变薄是由于上表皮、栅栏组织和海绵组织厚度减少；叶变小是由于上表皮细胞数量显著变少。在前期对SCL2（暂命名Cssl修改为 SCL2）精细定位基础上，利用 2500 株作图群体将 SCL2 基因定位在 Chr7 上 56.8 kb区间，内含10个候选基因，其中只有CsPHP在第5个外显子上存在一个非同义SNP位点，由此该基因是SCL2的候选基因，编码假定的核苷二磷酸磷酸酶，但在植物中其作用和功能未知。时空表达分析发现，候选基因在突变体与野生型之间并无转录水平的差异；对该基因进行拟南芥和烟草的遗传转化，但转化植株未有表型变化。由此对该候选基因存疑，重新进行精细定位。将作图群体扩大至8038株，将SCL2定位在12.07 kb区间内，全长测序、大群体的连锁分析及位点唯一性检测再次证实CsPHP是SCL2的候选基因。对该基因进行黄瓜的遗传转化，已获得杂合的阳性植株。CsPHP蛋白定位于细胞膜上。外源激素喷施和内源激素含量分析表明，内源激素并不直接参与突变体小叶形成。叶极性发育调控网络的关键基因如ARF4，AS1，AGO7，FIL，KAN1，YUC5，WOX1及PIN1-PIN6表达分析说明，突变体小叶形成与极性发育无直接关联。大肠杆菌互补试验证实候选基因与组氨酸的生物合成无关。利用HPLC分析叶片中能量状态，发现CsPHP可以将ADP水解为AMP，暗示能量稳态在突变体小叶中起重要作用。这是首次报道PHP蛋白在植物中的可能作用。突变体与野生型叶片光合参数无显著差异，通过dCAPS-C1245标记辅助筛选，将突变体小叶基因转入课题组的6份骨干种质中，已经获得携带小叶基因的BC4育种材料。