大豆低氮胁迫响应的GATA转录因子的功能解析

Nitrogen is an important macronutrients required for plant growth and a limiting factor for crop productivity.Soybean is a valuable oilcrop that provides protein and requires a large amount of nitrogen to accumulate high levels of nitrogen in the seed. Though soybean can acquire nitrogen via its nitrogen-fixing symbiosis with rhizobacteria,the large amount of nitrogen fertilizers are also applied to increase soybean productivity because the symbiosis has not completely established at the seedling stage and begun to decline at the ripening stage.The low efficient use of nitrogen fertilizer not only directly affects yields and quality of soybean products,but also causes enviromental pollution. Improving the nitrogen-use efficiency(NUE)is therefore crucial. At present, the NUE mechanism is unclear and the information on genes associated with NUE is lack in soybean. Solex sequencing results showed that a GATA transcription factor expression was significantly induced in a low-N-tolerant variety PoHuang under N-limited conditions. Many GATA factors have been shown to be involved in the global regulation of nitrogen metabolism in fungi. In this study, the ORF and promoter of the GATA transcription factor will be isolated.The molecular function of the GATA transcription factor involved in the high NUE will be estimated through subcellular analysis,overexpression and suppression of this gene in soybean,and compensary analysis in Arabidopsis mutant. This will provide a detailed understanding of NUE mechanisms and also provide a basis for breeding soybean varieties that are tolerant to low-N conditions.

氮作为植物生长发育所必需的大量元素，是影响植物产量的重要限制因子。大豆富含蛋白质，是高需氮的重要油料作物。尽管大豆可通过根瘤固氮,但在苗期和成熟期因根瘤固氮系统尚未完全建立或已开始衰亡，仍需要额外施用大量氮肥。而氮肥的低效利用不仅直接影响了大豆的产量和品质，还严重污染了环境。提高大豆氮利用效率是解决上述问题的根本途径。目前，大豆氮高效利用的分子机理还不十分清楚，相关的基因信息也十分有限。前期表达谱测序结果显示一个GATA转录因子在大豆耐低氮材料坡黄中受低氮显著上调表达。该类转录因子被认为广泛参与调控真菌氮代谢过程。本项目拟通过克隆GATA转录因子基因及启动子，对GATA转录因子进行亚细胞定位、超量表达、抑制表达、突变体互补等分析该基因参与大豆氮高效利用的分子功能，为揭示大豆氮高效利用分子机制和进行氮高效利用分子育种奠定基础。

氮是影响作物生长、发育和产量的关键限制因子之一。本项目对大豆中受硝酸盐响应的GATA转录因子进行了初步地鉴定与功能分析。亚细胞定位结果显示GmGATA转录因子定位于细胞核，组织特异性表达分析与启动子表达模式分析表明GmGATA转录因子主要在光合组织叶片中表达。AtGNC是大豆GATA转录因子的同源基因，可调控拟南芥碳氮代谢过程。将GmGATA超量表达载体转化拟南芥gnc突变体，可互补突变体的表型，转基因材料叶片叶绿素含量显著增加，揭示了GmGATA基因与AtGNC基因的生物学功能具有保守性。利用农杆菌介导的子叶节遗传转化方法将GmGATA超量表达载体转化天隆一号大豆，Bar基因试纸条和PCR检测共确定了四株阳性转化植株。QPCR分析发现GmGATA在其中两个株系GATAOX-1和GATAOX-2中的表达水平显著高于对照。进一步的表型观察发现转基因株系GATAOX-1和GATAOX-2下胚轴、茎、叶和嫩荚的颜色均呈深绿色，显著深于野生型。QPCR分析发现转基因株系中叶绿素合成MEP途径第一步酶基因GmDXS1和最后一步酶基因GmPOR1、GmPOR2的表达水平较野生型显著提高，揭示了GmGATA基因可调控大豆叶绿素的合成。叶绿素含量的高低常可反应植物体内氮素水平。转基因株系叶片中氮代谢相关基因的表达分析结果表明GmINR2和GmNIR的表达水平显著高于对照，而GmNRT1-2和GmGS1的表达水平显著低于对照，说明GmGATA基因可调控大豆氮代谢过程。此外，超量表达GmGATA基因可增加大豆种子的百粒重。上述结果揭示了GmGATA基因在调控大豆叶绿素合成和氮代谢过程中发挥着重要的作用，为大豆氮高效利用的分子机理研究奠定了基础。