氮高效型饲用香椿的筛选及其氮高效机制研究

Toona sinensis (A.Juss.) Roem (TS), whose buds are the traditional Chinese “forest vegetables” with outstanding health benefits, had been widely cultivated with the planting area over 1 million acres. But till now, the mature leaves of TS have not been utilized. Our previous researches have shown that the mature leaves of TS own prominent forage value, but they are also rich in nitrates. So low nitrogen cultivation is demanded. Nitrogen is important to the growth and protein accumulation of plants. Hence, screen out the genotypes of TS which can grow in the low nitrogen input condition with high quality output is the key for the development of feeding-exploitation of mature leaves of TS. By the analysis of the content of crud protein, flavonoids and nitrate and biomass, the presented study plan to seek out 20 kinds of feeding-suit germplasms among these 40 quality germplasms selected in the earlier investigation. Then, responses of selected genotypes of TS to different nitrogen concentration (0.1，0.5，1，5，10，15 mM) will be examined in their seedling and mature stages. The genotypes of TS with highest and lowest nitrogen us efficiency (NUE) will be picked out, and their stems、leaves and roots will be used to do RNA-seq. Genes which are significant to the regulation of NUE will be searched and compared by qRT-PCR. Finally, combine the results of morphological, physiological, biochemical and molecular biological analysis, this study aims to reveal the mechanism of the regulation of NUE of TS，and to lay the foundation for the further directive breeding of TS with biotechnology.

香椿(Toona sinensis (A.Juss.) Roem)，其嫩芽是我国传统特色“森林蔬菜”，保健功效突出，全国推广面积已超100万亩，但其大量的成熟老叶尚未得以利用。本团队前期研究表明，香椿老叶具有突出的饲用价值，但其易富集硝酸盐，需低氮栽培。氮对植物生长及蛋白积累有重要影响。因此，筛选出低氮条件下仍能优质高产的氮高效型香椿是其老叶饲用开发需解决的关键问题。本项目拟以调查所得40份优良香椿种质为材料，通过分析其老叶粗蛋白、类黄酮、硝酸盐含量及产量，初步筛得20份适合饲用的基因型；此后，考察其在苗期及成熟期对供氮量(0.1，0.5，1，5，10，15 mM)的响应，选出氮效最高和最低的饲用香椿基因型，并对其茎叶和根进行比较转录组分析，结合qRT-PCR选出氮效调节关键基因。最后，综合形态、生理生化及分子生物学分析结果，剖析香椿的氮高效机制，为今后利用生物技术定向改造其氮效奠定基础。

香椿（Toona sinensis （A. Juss） Roem）在我国具有悠久的食用栽培历史，其嫩芽风味独特，是享有盛誉的“森林蔬菜”；其成熟复叶营养丰富，具有饲用潜力。但是，香椿优良品种缺乏，市场种苗的生长特性及营养组成，特别是蛋白及硝酸盐积累差异显著，亟需筛选高氮效的香椿良种，并阐明其氮代谢调控的深层机制。本项目在对我国香椿资源进行全面调查和收集的基础上，系统分析了各种质的饲用品质和氮利用效率，并从生长性能、生理生化特性和分子调控特点方面剖析了氮高效型优势饲用香椿种质对不同氮供给水平（0.1 mM-15 mM）的响应机制。研究结果表明：1）随香椿复叶不断成熟，其粗蛋白含量逐渐降低；类黄酮含量逐渐升高，且其与抗氧化活性显著正相关；硝酸盐呈“V”形积累，7-8月适合进行饲用采收。2）不同香椿种质的氮含量、类黄酮组成、生物量及光合性能存在显著差异，综合评估并聚类后，10份高氮效、高生物量、高酚的氮高效型香椿种质聚为类群Ⅱ。3）对氮效极端差异的两种香椿种质进行比较分析，发现氮高效香椿对低氮及高氮环境有更好的适应性，其生物量及氮积累量均较高，类黄酮积累差异和光合特性差异可能是导致两类香椿氮效差异的重要原因。4）比较转录组分析表明，氮高效香椿中氮代谢相关基因（Nrt及Gln）的持续高表达是保证其高氮效的基础，而类黄酮代谢的灵活调节（低氮时CHS、F3’H、DFR、ANS、LAR、MYB及bHLH高表达，高氮时其表达下调）是其适应低氮及高氮环境的关键。基因共表达网络分析表明，部分丝氨酸/苏氨酸代谢、酚类代谢及光合相关基因与其它基因的关联度均较高，可能是连接相关分子调控网络的中枢基因。本项目研究结果将为香椿良种选育和氮效改良提供重要理论依据。