编码分泌型小肽的小麦低氮响应基因TaLNR1的功能分析

Low nitrogen stress can induce wheat root elongation for acquiring nitrogen in deep soil. However, we know very little about the molecular mechanism of it. Identification and functional analysis of regulatory genes of wheat root elongation induced by low nitrogen stress would be very meaningful for genetic improvement of wheat nitrogen use efficiency. In the former project supported by NSFC, we isolated a low nitrogen responsive gene and named it as TaLNR1, which encoding a small secreted peptide consists of 65 amino acid residues. Overexpression of this gene lead to a longer lateral root system compared to wild type in both normal and low nitrogen conditions. We proposed this project here to further investigate the function of TaLNR1 gene. The content of this project includes the following components: (1) To clone and perform expression analysis of different members of TaLNR1 gene corresponding to A, B and D genome in wheat. (2) To analysis the function of TaLNR1 gene by overexpressing and underexpressing it in wheat; (3) To investgate the phenotype of root trait of wheat plants after fed with synthetic small peptides in both modified or non-modified forms. (4) To measure the expression level of genes in regulatory pathways of root development and nitrogen use efficiency in wild type, overexpressing and underexpressing transgenic lines, and plants fed with synthetic peptides. This research will improve the understanding of the biological function and molecular mechanism of TaLNR1 gene.

低氮能诱导小麦根系伸长以增加其利用深层土壤中氮素的能力，挖掘相关调控基因对提高小麦氮素利用效率，选育氮高效新品种具有重要意义。申请人在青年基金资助下，发现一个受低氮诱导上调表达的未知功能基因，编码产物为包含65个氨基酸残基的分泌型小肽。在拟南芥中超表达后能显著增加侧根长，将其命名为TaLNR1。本项目拟在前期工作基础上，对其功能和作用机理开展进一步研究。拟首先克隆该基因对应于小麦A、B和D基因组的成员并进行遗传定位，明确其表达的主要成员和组织部位；进而通过在小麦中超量表达和RNAi减量表达该基因以及通过人工合成小肽进行根系饲喂试验深入研究TaLNR1基因的功能；最后，在野生型、超表达系和减量表达系以及根系饲喂试验处理前后的植株中，检测调控根系发育和氮素吸收利用相关基因的表达变化情况，对该基因的作用机理进行分析。以期通过上述研究解析TaLNR1基因的功能及其作用的分子机理。

氮素易随水分淋溶到土壤深层，而低氮能诱导小麦根系伸长以增加其利用深层土壤中氮素的能力。研究低氮诱导小麦根系伸长的分子机理对于减少氮肥投入，提高小麦氮素利用效率具有重要意义。本项目主要围绕课题组前期鉴定的一个编码分泌型小肽、受低氮诱导上调表达的功能未知基因（TaLNR1）开展工作。首先克隆了TaLNR1对应于小麦A、B和D组的成员，并分别定位于小麦7A、7B和7D染色体上，定量PCR结果表明TaLNR1主要在根部表达，且A组和D组成员为其主要成员。分别利用A组成员TaLNR1-A1的编码区序列和A、B和D组成员的保守序列构建了该基因的超表达系和RNAi减量表达系。结果表明，过量表达该基因能显著提高小麦的最长根长和总根长，而减量表达则降低了小麦的总根长，对最长根长没有明显影响；在低氮条件下，超表达系苗期地上部和根系的氮浓度均显著增加，成熟期秸秆氮浓度和籽粒氮浓度也显著增加。外源饲喂TaLNR1基因编码的小肽片段能增加拟南芥的侧根数、侧根长、主根长和总根长和总根长。定量PCR结果表明，TaLNR1可能主要是通过影响根系发育相关基因和硝酸根转运基因的表达调控了根系性状和氮素转运。本项目研究结果有助于进一步理解低氮诱导小麦根系伸长的分子机理，并为遗传改良小麦根系性状和提高氮素利用效率提供基因资源。