高亲和氮素转运蛋白在茶树低氮耐受中的作用机制

Nitrogen plays a crucial role on the production and quality of tea. Understanding the mechanisms of nitrate uptake from the soil in response to nitrogen deficiency is an important step on the way to breed the high nitrogen use efficiency (High-NUE) cultivars in tea, and to prevent negative effects of nitrogen fertilizers on the environment. It has been indicated that high-affinity nitrogen transporters play an essential role in model plants under low nitrogen condition. The high affinity nitrogen transport systems (HATS) acts at low NO3 concentrations and hence is important in N limited conditions. However, how much do HATS contribute to root NO3 uptake activities in nitrogen-deficiency tea plants? What’s the main component of HATS in tea roots under low nitrogen conditions and how does it function?. In our previous studies, 7 transcripts (3 CsAMT1 and 4 CsNRT2) belonging to HATS gene families have been found from tea root transcriptomic. Meanwhile, the full-length cDNA sequences of these genes have been cloned. Here, two bulked populations of tea are generated from F1 progeny showing contrasting phenotype in nitrogen use efficiency under N-deficiency conditions, and the root transcriptomic of two bulked samples under N-deficiency conditions will be analyzed by RNA-seq. Combined with N uptake efficiency determined by Continuous Flow Analytical System(CFA), Bulked-segregant analysis (BSA) will be applied to identify some essential HATS genes associated with N uptake under low nitrogen conditions. Furthermore, the tissue expression patterns and the temporal expression patterns in response to low nitrogen conditions of identified HATS genes will be analyzed by QT-PCR. Finally, transgenic Arabidopsis will be established to verify their function. By combining correlation analysis and gene co-expression network analysis, A gene network for regulating the NUE will be constructed. We will uncover the regulation mechanism of NUE under low nitrogen condition in tea.

氮素对茶树产量与品质具有十分重要的作用。研究低氮胁迫下茶树氮素吸收的机制，对氮高效茶树培育及防止茶园氮肥施用带来的环境问题，都有着重要的作用。模式植物中的研究表明，高亲和氮素转运蛋白（High Affinity Transport Systems, HATS）在缺氮条件下的氮素吸收中发挥了重要作用。HATS对低氮条件下茶树氮素吸收有多大贡献？低氮条件下，起作用的主要的HATS组分及其作用机制又是什么呢？课题组前期，已克隆得到7个HATS基因的全长cDNA序列。在此基础上，本课题以两组氮效率截然不同的茶树F1子代个体为试验材料，采用集群分离分析（BSA）原理，分析二组氮效率差异群体在低氮胁迫下的转录表达差异，结合氮吸收效率值找出与茶树低氮下氮吸收有关的HATS基因，明确HATS基因对低氮胁迫的响应及组织表达特性，转基因拟南芥验证其功能，构建基因调控网络，揭示茶树低氮环境下氮效率调控机制。

茶树是氮肥需求量较高的作物，而无机氮肥的施加，会使茶树根系释放大量的H+，造成了茶园土壤急速酸化。研究茶树对无机氮素离子的吸收利用机理，对减少茶园氮肥施用量，提升氮素利用率等都有重要的指导意义。本文通过氮素处理条件下茶苗根系转录组测序，采用WGCNA分析构建茶树氮代谢网络，结合氮素吸收基因在不同氮素处理的表达模式和转基因功能验证，探讨茶树氮素代谢的分子机制。. 本研究共筛选到收氮素浓度调控的差异表达基因4587个，并利用WGCNA分析构建了初级氮代谢调控网络，在网络中Degree ≥ 10的基因，包括铵根转运蛋白家族成员（AMT1.2）、硝酸根转运蛋白家族成员（NRT2.4）等；克隆得到茶树铵根转运蛋白基因3个（CsAMT1.1，CsAMT1.2，CsAMT3.1）、茶树硝酸根转运蛋白基因7个（CsNRT2.4、CsNRT2.5、CsNRT3.1和CsNRT3.2等）和茶树铁氧还蛋白亚硝酸还原酶基因CsF-NiR，其中在根系中受到氮素诱导而表达量明显上调的基因为CsAMT1.2、CsNRT2.4、CsNRT3.2和CsF-NiR；采用转基因方法对CsAMT1.2、CsNRT2.4和CsF-NiR进行基因功能验证，研究表明，CsAMT1.2为质膜蛋白，可以补偿铵根吸收缺陷型酵母31019b在低氮下的铵根转运能力，并提升了31019b酵母对低浓度铵根离子（0.1 mM NH4+）的吸收速率。CsNRT2.4同样为质膜转运蛋白，在0 和0.1 mM NO3- 条件下可以促进拟南芥的侧根发育和生物量积累，且可以提高野生型拟南芥在低硝酸根（0.1 mM NO3-）浓度下的吸收速率。CsF-NiR定位在细胞膜及叶绿体中，且可以提高拟南芥AtNiR1基因缺失突变体的叶绿素含量。.本研究揭示CsAMT1.2为高亲和性铵根转运蛋白，负责低氮条件下茶树根系对土壤中NH4+的主动吸收；CsNRT2.4为高亲和性的硝酸根转运蛋白，是茶树根系NO3-吸收的关键基因；而CsF-NiR在茶树根及叶片的亚硝酸还原过程中均发挥了重要作用。