拟南芥细胞质谷氨酰胺合成酶家族各成员在种子萌发过程中的功能研究

As a key enzyme in plant nitrogen metabolism, cytosolic glutamine synthetase（GS1）plays an important role for plant growth and development. In Arabidopsis, GS1 is encoded by 5 isogenes (AtGln1;1-5). This project explores specific functions of the individual GS1 isogenes in Arabidopsis during seed germination, by using molecular biology, biochemistry, plant physiology and advanced microscopy techniques. With respect to an essential Arabidopsis GS1 isogene — AtGln1;2, experiments of characterizing AtGln1;2-knockout mutant and altering exogenous nitrogen supply will be performed to investigate its molecular mechanism in regulating seed germination. Real-time tissue and cellular localizations of different AtGS1 isogenes will be dynamically observed by novelly developed microscopy during the whole germination period. In addition, the specific functions of AtGS1 isogenes in nitrogen assimilation will be studied by characterizing their knockout mutants. Moreover, changing external nitrogen during seed germination will help to uncover responses of important AtGS1 isogenes in regulating nitrogen assimilation at the very early growth stage, thus better understand their functions. This project is studying the functions of the individual AtGS1 isogenes during seed germination, which helps to further explore the molecular mechanisms in plant nitrogen assimilation, thus provides useful theories for molecular breeding.

作为植物氮代谢中的关键酶，细胞质谷氨酰胺合成酶(Cytosolic Glutamine Synthetase，GS1)在植物的生长发育中起重要作用。在拟南芥中，GS1家族包含5个亚成员(AtGln1;1-5)。本项目将利用分子生物学技术、生理生化研究手段及自研发的高级显微成像技术，对拟南芥GS1家族各成员在种子萌发阶段的功能展开探索。针对主要的氮同化基因 — AtGln1;2，将通过改变其表达水平和外源氮供给实验，深入研究该基因调控种子萌发的机制。本项目将实时监测拟南芥种子萌发过程中GS1家族不同成员的组织及细胞水平的动态定位和表达情况。同时研究GS1家族成员的缺失，对种子萌发阶段氮同化的影响。在此基础上，研究在种子萌发时期起主要作用的GS1家族成员对外源氮供给的响应情况。本项目研究拟南芥种子萌发过程中GS1家族各成员的功能，有助于了解种子萌发阶段氮代谢的机制，并为植物育种提供理论基础。

种子萌发开启了植物新一代生命的延续。可是，由于研究手段的限制，该过程远未被认识清楚。申请人在前期工作中发现：拟南芥细胞质谷氨酰胺合成酶（AtGS1）基因家族的五个成员功能各异，通过相互协作共同调控植物整个生长周期的氮（N）代谢。但是，种子萌发阶段的N同化分子机制，还是未解之谜。本项目采用高级显微成像技术，寻找种子萌发阶段调控N同化的关键基因。结合荧光定量PCR检测结果，找到了表达量较高的AtGln1;2。提出假说：AtGln1;2可能是种子萌发时期调控N同化的重要基因。对AtGS1家族主要基因（AtGln1;1-3）的敲除突变体研究进一步验证了AtGln1;2在调控种子萌发时期N同化和N运输过程中，发挥了重要的作用。尤其在铵处理环境下，AtGln1;2甚至可以通过表达上调来弥补家族中其他成员的功能缺失。借助经典遗传学、分子生物学、超高分辨率显微成像等技术方法，着重揭示了植物生长发育早期体内N同化的分子机制。