黄瓜转录因子CsHY5响应弱光胁迫调控硝酸盐吸收与分配的分子机制研究

Cucumber is one of the main vegetable cultivated in greenhouse in China, which is often subjected to weak light stress during winter season, and leading to a decreased yield. In addition to the damage to the photosynthetic system of leaves, weak light also inhibited the assimilation and metabolism of nitrate (NO3-), but the regulatory mechanism is still unclear. Previous studies have shown that bZIP transcription factor HY5 plays an important role in the light signal pathway, which is involved in the regulation of plant growth and development such as light morphogenesis, stress resistance and mineral element absorption. In the preliminary study, we isolated a putative cucumber CsHY5 gene by homologous comparison. Based on the subcellular localization of CsHY5 and its expression profile in response to light and NO3-, this project intends to verify the activation activity of CsHY5 on the high affinity NO3- transporter family gene (CsNRT2) of cucumber by EMSA and yeast single hybridization; the CsHY5 overexpression and the gene editing vector of CRISPR-Cas9 knockout vector were constructed and transformed into cucumber, then the ability of transport of NO3- in wild-type and transgenic plants were compared under weak light stress, and the expression of CsNRT2 family was also analyzed. Through the above research, the aim of our project was to identify the molecular mechanism of transcription factor CsHY5 improving the NO3- metabolism by regulating the CsNRT2 family in response to light, and providing a theoretical basis for regulating of CsHY5 to enhance cucumber resistance to weak light stress.

黄瓜是我国设施栽培的主要蔬菜之一，冬季栽培时常遭受弱光胁迫而抑制生长、降低产量。除了降低叶片的光合能力外，弱光还抑制了黄瓜对硝酸盐（NO3-）的同化代谢，但其调控机制尚不明确。已有的研究表明，bZIP类转录因子HY5在光信号通路中具有重要作用。我们前期克隆得到一个假定的黄瓜转录因子CsHY5。在初步探明CsHY5亚细胞定位及其表达模式对光照和NO3-响应的基础上，本项目拟通过EMSA和酵母单杂交验证CsHY5对黄瓜高亲和NO3-转运蛋白家族基因（CsNRT2）的激活活性；构建CsHY5的过表达和CRISPR-Cas9基因编辑敲除载体并转化黄瓜，比较野生型和转基因植株弱光胁迫下对NO3-的转运能力；分析CsHY5对下游CsNRT2家族基因表达的影响。通过以上研究，揭示CsHY5响应光照后调控CsNRT2家族表达改善自身NO3-代谢的分子机制，为通过调控CsHY5增强黄瓜耐弱光性提供理论依据。

本研究首先利用LED灯的窄光谱特征，以白光为对照，验证了在低氮环境下生长的黄瓜对单色红光和单色蓝光的影响特征，证明蓝光可以通过提高黄瓜在低氮环境下的光合作用、根系发育和改善氮代谢通路，进而提高其对低氮环境的耐受性；其次，通过前期试验发现蓝光可能通过调节转录因子CsHY5来响应低氮胁迫，因此我们克隆了黄瓜CsHY5基因，检测了其表达模式和定位，并利用DNA亲和纯化测序技术筛选了CsHY5调控的下游基因，结合基因表达量和基因启动子结合位点，确定了CsHY5能够正向调控CsNRT2.1和CsNIA2，初步明确了CsHY5在黄瓜幼苗响应蓝光提高低氮耐受性中的作用机制。