BnaNRT1.5s介导硝酸盐转运调控油菜氮素利用效率

BnaNRT1.5s mediates nitrate transporter to regulate nitrogen use efficiency in Brassica napus

摘要:

提高油菜氮素利用效率(nitrogen use efficiency,NUE)对于实现我国“化肥零增长”目标及保障油菜产业绿色发展具有重要意义。本研究旨在探讨硝酸盐转运蛋白基因BnaNRT1.5s对油菜氮素转运、利用效率的影响，为创制氮高效油菜提供优异的基因资源。通过对甘蓝型油菜‘Westar’不同生长时期、组织取样进行实时荧光定量PCR (quantitative real-time polymerase chain reaction,qRT-PCR)实验，探究氮素响应核心基因BnaA05.NRT1.5的时空表达谱，并利用亚细胞定位技术探究其在细胞中的表达模式；利用CRISPR/Cas9构建油菜BnaNRT1.5s敲除材料，并进行高氮(12.0 mmol/L)和低氮(0.3 mmol/L)处理的水培试验。幼苗培养21 d后，分别采集根和地上部样品，称重、测定氮含量、木质部伤流液硝酸盐含量并计算总氮和NUE。油菜硝酸盐转运蛋白BnaA05.NRT1.5定位于细胞膜。在苗期和蕾薹期，BnaA05.NRT1.5主要在根中表达；而在生殖生长期，其在老叶、成熟角果皮中表达水平较高。与野生型相比，在高氮和低氮处理下BnaNRT1.5s突变后幼苗植株干重和总氮都显著增加；在低氮条件下，根的NUE显著提高；在高氮和低氮条件下，地上部硝酸盐含量显著降低、根部硝酸盐含量显著增加，导致地上部与根部硝酸盐含量比值显著下降。BnaNRT1.5s参与调控硝酸盐从根部到地上部的转运，BnaNRT1.5s突变后提高了油菜幼苗的氮素吸收和利用，促进幼苗生长。本研究不仅为深入解析BnaNRT1.5s调控油菜NUE的生理分子机制提供了重要参考，也为油菜NUE的遗传改良提供了宝贵的基因资源。

Abstract:

Improving the nitrogen use efficiency (NUE) of Brassica napus is of significant importance for achieving the national goal of zero growth in chemical fertilizer application and ensuring the green development of the rapeseed industry. This study aims to explore the effects of the nitrate transporter gene BnaNRT1.5s on the nitrogen transport and NUE of B.napus, providing excellent genetic resources for the development of nitrogen-efficient B.napus varieties. The spatiotemporal expression of BnaA05.NRT1.5 as a key nitrogen responsive gene was profiled by qRT-PCR at different growth stages and for different tissue samples of B.napus ‘Westar’. Subcellular localization was employed to examine its expression pattern in the cells. Additionally, CRISPR/Cas9 was used to create BnaNRT1.5s knockout lines, which were subjected to hydroponic experiments under high nitrogen (12.0 mmol/L) and low nitrogen (0.3 mmol/L) conditions. After the seedlings were cultivated for 21 days, root and shoot samples were collected for weighing, nitrogen content determination, xylem sap nitrate content assessment, and calculation of total nitrogen and NUE. The B.napus nitrate transporter BnaA05.NRT1.5 was localized to the cell membrane. During the seedling and early bolting stages, BnaA05.NRT1.5 was predominantly expressed in roots, while it was highly expressed in old leaves and mature silique skin during the reproductive stage. Compared with the wild type, the mutant BnaNRT1.5s showed significant increases in the dry weight and total nitrogen of seedlings under both high and low nitrogen conditions. Under low nitrogen conditions, NUE in the roots of BnaNRT1.5s significantly improved. Notably, under both high and low nitrogen conditions, the nitrate content in the shoots of BnaNRT1.5s decreased significantly, while that in the roots increased significantly, resulting in a significantly decreased shoot-to-root nitrate content ratio. BnaNRT1.5s is involved in regulating the transport of nitrate from the roots to the shoots, and its mutation enhances nitrogen absorption and utilization in B.napus seedlings, promoting seedling growth. This study not only provides references for understanding the physiological and molecular mechanisms by which BnaNRT1.5s regulates NUE but also offers valuable genetic resources for improving NUE in B.napus.