大豆NAC转录因子SIN调控根瘤衰老的机制研究

Soybean is the main source of plant protein and vegetable oil. Rhizobia-soybean nodule symbiosis provides most nitrogen for soybean growth and development. However, the reduction of nitrogen fixation efficiency in senescent nodule could not meet the nitrogen demand in the later stage of soybean growth. Therefore, it is an effective way to improve the nitrogen fixation efficiency and yield of soybean to delay nodule senescence and prolong the time of nitrogen fixation. NAC transcription factors are widely involved in regulating plant senescence process. Exposing the biological function and regulation mechanism of NAC transcription factors in nodule senescence provides a new strategy for delaying nodule senescence .the mechanism of nodule senescence. In our previous study, two significantly up-regulated NAC genes SINa/b have been identified in senescent nodules. Overexpression of SIN can promote nodule senescence, while knockout of SIN can delay nodule senescence to improve the nitrogen fixation ability. This project intends to carry out research on this basis: 1) The target genes of SIN were identified by ChIP-seq and transcriptome analysis; 2) The function of target genes in soybean nodule during senescence will be studied by analyzing gene expression patterns, CRISPR knockout mutant and nodule specific overexpression plants phenotypes. The results will explain the molecular mechanism of SIN in regulating the senescence of soybean root nodules and provide an excellent candidate gene and theoretical basis for cultivating soybean with high nitrogen fixation efficiency.

大豆是植物蛋白和植物油的主要来源。根瘤菌-大豆根瘤共生固氮为大豆生长发育提供绝大部分氮素，但是大豆后期根瘤衰老导致固氮效率降低而不能满足大豆后期的氮需求。因此，适当延缓根瘤衰老、延长固氮时间，是提高大豆固氮效率和增加产量的有效途径。NAC转录因子广泛参与调控植物衰老过程，研究NAC转录因子在大豆根瘤衰老中的功能和调控机制，为延缓根瘤衰老提供新策略。本项目前期工作鉴定了两个在衰老根瘤中显著上调表达的NAC转录因子SINa/b，其超表达促进根瘤衰老，而敲除则延缓根瘤衰老并提高根瘤的固氮能力。本项目拟在此基础上展开研究：1）利用ChIP-Seq和转录组分析等鉴定SIN的靶基因; 2）通过基因表达模式分析、CRISPR敲除突变体和根瘤特异超表达表型研究基因在大豆根瘤衰老中的功能。研究结果将解析SIN调控大豆根瘤衰老的分子机制，为培育高效固氮大豆提供优良候选基因和理论基础。

大豆根瘤是大豆生长、发育、生产和种子品质的主要氮素来源。共生固氮是有时间限制的，因为根瘤在植物发育的生殖阶段特别是在种子发育期间衰老。根瘤衰老的特征是诱导衰老相关基因表达，如木瓜蛋白酶样半胱氨酸蛋白酶(CYPs)，最终导致根瘤菌和植物细胞的降解。然而，大豆根瘤衰老相关基因是如何被激活的尚不清楚。在本项目中，我们鉴定了两个同源的NAC转录因子GmNAC039和GmNAC018，是根瘤衰老的主要调控子。超表达两者任何一个都会导致根瘤衰老，而敲除它们可以延缓衰老并增加大豆根瘤的固氮酶活性。转录组分析和nCUT&Tag-qPCR检测显示，GmNAC039直接与核心基序CAC(A)A结合，激活4个GmCYP基因(GmCYP35/37/39/45)的表达。与GmNAC039和GmNAC018的观察结果相似，GmCYP基因在根瘤中的超表达或敲除分别导致早熟或延迟衰老。这些数据为了解GmNAC039/GmNAC018通过直接激活GmCYP基因表达调控根瘤衰老的调控机制提供了重要的见解。