CsGA20ox1在CsGL1途径中调控黄瓜果刺发育的分子机制

Warty fruit is highly valuable external quality trait, which is related to the market values of cucumber. However, little is known about molecular mechanism of the formation of fruit spines and tumors in cucumber, limiting the application of molecular marker-assisted selection and molecular design breeding for warty fruit trait. Our previous work indicated that CsGA20ox1 in cucumber was a homologous gene of a key gene for fiber development, GhGA20ox1, in cotton. In addition, a loss-of-function mutation of CsGL1, a key gene for fruit spine and tumore formation, significantly increased the expression level of CsGA20ox1. Therefore, CsGA20ox1 was likely regulated by CsGL1 and played an important role in the development of fruit spines. In the present study, over-expression, silence, spatial expression pattern and subcellular distribution of CsGA20ox1 will be performed to analyze its biological function. Moreover, transient expression, yeast one-hybrid and EMSA will also be used to analyze the relationships of CsGA20ox1 and its upstream gene with CsGL1. Then the molecular mechanism of CsGA20ox1 in the CsGL1 pathway regulating development of fruit spines will be elucidated preliminarily. Therefore, this project will not only provide the theoretical basis of molecular marker-assisted selection and molecular design breeding for warty fruit trait, but also enrich the theory of plant cell differentiation.

果实刺瘤是黄瓜重要的外观商品性状。然而，黄瓜果刺和果瘤形成的分子机制仍不清楚，限制了黄瓜刺瘤性状的分子标记辅助选择育种和分子设计育种的应用。本课题组前期的工作证明，黄瓜CsGA20ox1是赤霉素生物合成的关键基因且是棉纤维发育调控基因GhGA20ox1的同源基因。黄瓜果实刺瘤形成关键基因CsGL1功能的缺失使CsGA20ox1的表达量显著升高。因此，CsGA20ox1很可能受CsGL1的调控且在黄瓜果刺发育过程中起着非常重要的作用。本课题拟通过CsGA20ox1的过量表达、沉默、组织定位和亚细胞定位技术分析其生物学功能，并结合瞬时表达、酵母单杂交和EMSA等技术分析CsGA20ox1及其上游基因与CsGL1的关系，初步揭示CsGA20ox1在CsGL1途径中调控黄瓜果刺发育的分子机制。本课题不仅为黄瓜刺瘤性状的分子标记辅助育种和分子设计育种提供理论依据，还可以丰富植物细胞分化理论。

果实刺瘤是黄瓜重要的外观商品性状。然而，黄瓜果刺和果瘤形成的分子机制仍不清楚，限制了黄瓜刺瘤性状的分子标记辅助选择育种和分子设计育种的应用。本课题主要通过CsGA20ox1的过量表达、沉默、组织定位和亚细胞定位技术分析其生物学功能，并结合瞬时表达、酵母单杂交和EMSA等技术分析CsGA20ox1及其上游基因与CsGL1的关系，旨在探究CsGA20ox1在CsGL1途径中调控黄瓜果刺发育的分子机制。结果显示，CsGA20ox1具有负调控黄瓜表皮毛和果刺发育的功能。组织特异性分析表明，CsGA20ox1在子房、叶片、根和茎中的表达量较高，在其他组织中的表达较低。亚细胞定位结果表明，CsGA20ox1-GFP融合蛋白在烟草叶片表皮细胞的细胞核和细胞质中均有荧光信号。利用酵母单杂交筛库没有筛选到与CsGA20ox1启动子结合的互作蛋白，但我们利用植物转录因子数据库PlantTFDB并结合MEME和FIMO筛选到2个可能与CsGA20ox1启动子结合的候选BBR/BPC转录因子Csa2G365700和GRAS转录因子Csa5G569350。利用GA3处理csgl1及‘大青把’幼苗，结果表明，CsGA20ox1的表达量在csgl1及‘大青把’幼叶中的表达量均下调，但二者之间并无显著性差异。利用烟草瞬时表达系统没有发现CsGL1能够直接调控CsGA20ox1的表达。酵母单杂交也没有检测到CsGL1与CsGA20ox1启动子的相互作用。结果表明，CsGL1与CsGA20ox1之间可能还有一些基因参与黄瓜表皮毛和果实刺瘤的发育途径或二者在两条相互独立的途径中，具体还有待进一步研究。