百合LoAMS转录因子调控花药绒毡层发育的功能与分子机制解析

Lily possesses an elegant flower shape and a beautiful implicit meaning. It is one of the most economic cut flowers worldwide. The lily’s anther is in a large size with enriched pollens. However, once the anther splits, those pollens can stain surrounding surfaces and consumers’ clothes easily, and stained marks are very difficult to be removed. Therefore, the pollen staining largely affects its economic values and usages. The genetic engineering technic is an effective way to solve the pollen staining problem by modifying the anther development specifically without changing other good plant’s traits. Based on our morphological and histological analysis of the anther development in the popular cultivar ‘Siberia’, an essential tapetum developmental related transcription factor, LoAMS, was cloned. Currently, although we have analyzed its expression patterns, protein characteristics and the function on the anther development, the regulation mechanism of LoAMS is still largely unknown. In this study, the function of LoAMS is further investigated. To explore upstream regulators of LoAMS, yeast one-hybrid screening is used with lily cDNA library; to characterize the upstream regulator gene, the gene silencing and the ectopic expression assays are performed; to detect the DNA-protein interaction and the regulation pattern of the upstream regulator on LoAMS, EMSA, yeast one-hybrid and the Dual-Luciferase assay are employed. Taken together, this research is helpful to understand the molecular mechanism of LoAMS in regulating the anther development. Meanwhile, this work is able to provide some candidate genes and theoretical information for the breeding of new lily cultivars without pollen pollution using genetic engineering.

百合花型优美，寓意美好，是重要的鲜切花，但百合花药大、花粉量多，若不及时去雄，散落的花粉会污染周围环境和消费者衣物，严重影响观赏和应用价值。如何在不改变百合其它优良性状的基础上，通过基因工程定向改变花药发育，解决花粉污染问题是提高百合观赏和应用价值的一个重要途径。课题组在对主栽百合品种‘西伯利亚’花药组织切片观察的基础上，从中克隆调控绒毡层发育的关键转录因子LoAMS，并进行了初步基因表达分析、蛋白特性分析及功能验证，但具体的作用机制并不清晰。本项目拟进一步验证LoAMS基因功能，利用酵母单杂筛选其上游调控转录因子，通过基因沉默、拟南芥中异位表达验证基因功能，凝胶迁移、酵母单杂、双荧光素酶试验检测上游转录因子与LoAMS启动子的结合性与调控转录模式，从而进一步解析LoAMS调控花药发育的作用机制，为通过基因工程改良百合花粉污染问题提供候选基因和奠定理论基础。

百合是优良的鲜切花，在世界切花市场占有重要位置。单瓣百合花型优美，但花药较大，若不及时去雄，散落的花粉会污染花瓣和消费者的衣物，影响观赏和应用价值。本研究以“西伯利亚”百合为试验材料，对不同发育时期的花药进行了切片观察；从3cm花蕾的花药中分离得到LoAMS基因，ORF全长1626bp，编码541个氨基酸，属于MYC家族蛋白，编码的蛋白定位于细胞核中，属于转录激活因子，转录激活域位于C端的204个氨基酸内。LoAMS基因在3cm，10cm花蕾的花药中相对表达量较高，推测该基因在百合花粉母细胞减数分裂后及花粉包被形成时期发挥重要作用。将LoAMS基因在百合花蕾中进行瞬时沉默，互补拟南芥ams突变体，验证了该基因调控百合花药发育的功能。根据切片结果，选取花药发育的3个关键时期：四分体时期，独立小孢子形成时期，花粉粒成熟期，进行了转录组测序，对转录组数据进行了分析，从中筛选出调控花药发育的关键基因。对LoAMS进行了互作蛋白筛选，上游转录因子筛选，初步筛选出了部分互作蛋白。克隆了LoAMS上游转录因子LoTDF1，ORF全长936bp，编码311个氨基酸，属于MYB家族转录因子，LoTDF1蛋白定位于细胞核中，LoTDF1在2cm花蕾中花药的相对表达量最高，推测LoTDF1在花粉母细胞阶段参与调控花药发育；克隆得到半胱氨酸蛋白酶LoCP基因，ORF全长1062bp，共编码353个氨基酸，LoCP基因在8cm花蕾的花药中相对表达量最高，其次为6cm花蕾中的花药，推测LoCP调控了百合花药绒毡层细胞的适时降解。本研究进一步解析了LoAMS基因调控花药发育的作用机制，研究了LoAMS上游转录因子LoTDF1和调控绒毡层适时降解的关键基因LoCP，实验结果为通过基因工程手段定向改变百合花药发育，培育无花粉污染品种，提供了理论基础与候选基因，对百合切花生产和销售，具有重要的理论和实践意义。