NRT基因对不结球白菜硝酸盐含量的影响及作用机理

Non-heading Chinese cabbage (Brassica campestris L. ssp. chinensis Makino; syn. B. rapa L), originated from China, is one of the vegetable crops with the largest sowing area in China, and it prefers nitrate nitrogen fertilizer. In order to improve the yield of non-heading Chinese cabbage, excessive fertilizers were applied in recent years which led to high nitrate concentration in plant tissues. Excessive nitrate application caused the environmental pollution and at the same time posed a potential risk to human health. In this project, two genes encoding nitrate transporter BcNRT1.1 and BcNRT2.2, were characterized through Real-time PCR analysis, Northern hybridization and subcellular localization. On this basis, by constructing a gene overexpression or RNAi interference vector, the genes were transformed into Chinese cabbage. The regulatory mechanism of BcNRT1.1 and BcNRT2.2 for accumulation of nitrate in non-heading Chinese cabbage was comprehensively investigated. And a further application of microarray techniques to analysis gene expression differences in nitrate uptake process of transgenic and non-transformed plants was carried out, to explore the gene biological function and potential breeding values in geoponics. This study aims to reveal the effect of nitrate transporter gene on nitrate concentration in non-heading Chinese cabbage and its biological functions, to lay the foundation for breeding new Chinese cabbage varieties with high nitrogen utilization efficiency and low nitrate content by means of molecular biology techniques.

不结球白菜(Brassica campestris L. ssp. chinensis Makino)属十字花科芸薹属芸薹种白菜亚种，原产我国，是中国蔬菜作物中栽培面积最大的一种，而且是喜硝态氮肥的叶菜类蔬菜。近年来菜农为了提高产量大量偏施氮肥，使不结球白菜硝酸盐含量超标，环境受到污染的同时给人民健康带来潜在的威胁。本项目在已经获得的两个编码硝酸盐转运蛋白的基因BcNRT1.1和BcNRT2.2的基础上，通过Real-time PCR分析，Northern杂交和亚细胞定位了解基因表达特性，在此基础上通过构建基因过量表达或RNAi 干涉载体转化不结球白菜，全面解析BcNRT1.1和BcNRT2.2基因表达对不结球白菜硝酸盐累积的调控机制。并进一步应用基因芯片技术，分析转基因植株和非转化植株硝酸盐吸收过程中的基因表达差异，探讨基因生物学功能及其在农艺、品质性状方面潜在的育种价值。

不结球白菜属十字花科芸薹属芸薹种白菜亚种，原产我国，是喜硝态氮肥的叶菜类蔬菜。硝酸盐，作为主要的植物氮素吸收形态，主要通过HATS和LATS两种系统进行运输。本研究克隆了不结球白菜的硝酸盐转运蛋白基因BcNRT1.1、BcNRT2.1以及BcNRT2.2。BcNRT1.1全长773bp。BcNRT1.1优先在植物根尖和地上部生长点表达。在拟南芥突变体chl1-5中过量表达BcNRT1.1，硝酸盐处理0.5h后，NRT2.1基因表达水平比突变体显著提高，表明BcNRT1.1通过调节NRT2.1基因表达参与初期硝酸盐反应。BcNRT2.1全长1789bp，编码530个氨基酸。氨基酸序列与拟南芥和甘蓝型油菜具有较高同源性。BcNRT2.1定位于细胞膜上。实时定量分析表明，BcNRT2.1主要在根部表达，受硝酸盐诱导。高浓度硝酸盐对BcNRT2.1诱导效果比低浓度硝酸盐更为强烈，但是高浓度硝酸盐的反馈抑制作用也更为明显。BcNRT2.2全长1593bp，编码530个氨基酸。氨基酸序列与拟南芥NRT2.2的同源性为86%。实时定量PCR分析结果表明: BcNRT2.2主要在根部表达，并受高浓度硝酸盐的诱导;在原始叶片中表达量较低，但受低温、高温和干旱胁迫的诱导后而高量表达。构建pCAM-NRT1.1和pCAM-NRT2.2 植物过量表达载体，采用Gateway试剂盒构建NRT1.1和NRT2.2的基因沉默载体，通过根癌农杆菌介导法转化不结球白菜品种‘苏州青’，经卡那霉素筛选，获得了抗性转基因植株。经PCR检测和GUS组织化学染色法证明抗性植株中整合了NRT1.1和NRT2.2基因。证明分别获得了4种载体的目的植株。分别用高浓度和低浓度硝酸盐溶液处理不结球白菜，其根和叶的差异表达基因具有不同的调控趋势。低浓度硝酸盐，两个组织中的差异表达基因一致的上调或下调表达，在高浓度硝酸盐下，显示相反的调控趋势。此外，功能注释发现在根中许多差异基因与水解酶活性、 转移酶活性、 核酸绑定转录因子活性相关。路径和网络分析表明不结球白菜硝酸盐代谢、运输和根系发育的具有整体效应。此外，实时荧光定量PCR分析表明了硝酸盐和干旱之间也存在关系。总的来说，我们发现大量的与不结球白菜硝酸盐转运和代谢相关的基因，这些发现对于深入研究不结球白菜代谢途径以及分子育种具有重要的意义。