大豆E3泛素连接酶基因GmPUB2在抗逆中的作用机制研究

GmPUB2 (Glycine max plant U-box 2, GmPUB2), encoding a U-box domain-containing E3 ligase protein, was induced by soybean mosaic virus (SMV), cold and salt stresses etc. The transgenic Arabidopsis plants overexpressing GmPUB2 demonstrated that GmPUB2 was dramatically upregulated by cold, drought stresses and exogenous hormone gibberellic acid (GA), salicylic acid (SA) compared to the wild types. In addition, overexpression of GmPUB2 in Arabidopsis resulted in an enhancement of drought stress tolerance, which may be regulated by ubiquitin-26S proteasome-mediated pathway. In order to clarify the mechanism of GmPUB2 in regulating stress responses, we will focus on the following researches. Overexpression and knock out strategy of GmPUB2 using Gateway and CRISPR/Cas9 technology and so on, were applied to dissect the function and signal pathway involved in soybean resistance to abiotic and biotic stress. The upstream and downstream proteins of GmPUB2 will be identified based on the iTRAQ (isobaric tags for relative and absolute quantitation, iTRAQ) data. Yeast two-hybrid assay and in vitro/vivo ubiquitination assays will be employed to identify GmPUB2 interacting proteins and substrates. The activity of 26S proteasome and the accumulation of ubiquitinated proteins were determined in GmPUB2 transgenic plants. As a result, we hope to clarify the function and mechanism of GmPUB2 by doing so, which may provide the theoretical basis for soybean breeding in biotic and abiotic stresses resistance.

本研究前期鉴定了一个参与大豆对SMV、冷害、盐害等胁迫响应的基因GmPUB2，其编码蛋白是含U-box结构域的E3泛素连接酶。转基因拟南芥研究表明GmPUB2受冷害、干旱和外源激素GA、SA等诱导上调表达，能显著提高拟南芥的抗旱性，推测其可能通过26S-蛋白酶体途径参与胁迫响应。为了阐明GmPUB2的抗逆机制，本项目拟在已有研究基础上，利用基因过表达和基因敲除等技术研究GmPUB2的功能及其参与的信号通路，分析GmPUB2调控大豆胁迫抗性的生理基础；根据蛋白组学数据鉴定GmPUB2调节的上下游蛋白并分析其可能的生物学功能；利用酵母双杂交筛选GmPUB2互作蛋白，鉴定GmPUB2的泛素化修饰底物并分析其生物学功能；分析GmPUB2转基因植株中蛋白酶体活性及泛素化蛋白含量的差异。综合所有结果，揭示GmPUB2在调控大豆抗生物及非生物胁迫过程中的功能及作用机制，为大豆抗逆遗传育种提供理论依据。

本项目研究了大豆GmPUB2基因的序列特征、表达水平、作用功能及抗逆机制等。序列分析发现该基因编码的蛋白是含U-box结构域的E3泛素连接酶。实时荧光定量PCR结果表明GmPUB2基因在大白麻的根、茎、叶、花、荚、籽粒中均有不同程度的表达，茎中的表达量最高。亚细胞定位结果表明该基因定位于细胞核上。转基因拟南芥研究发现GmPUB2受冷害、干旱和外源激素GA、SA等诱导上调表达，能显著提高拟南芥的抗旱性。NaCl处理发现过表达转基因大豆植株与对照相比，对盐害处理变得更敏感。通过基因编辑技术研究发现，突变型的大豆比野生型大豆更耐盐，但更感大豆花叶病毒。此外，运用酵母双杂交技术鉴定出8与GMPUB2互作蛋白。GST-pull down和BiFC技术验证了GMPUB2的互作蛋白。通过Co-IP的方法鉴定出Gm240400为GmPUB2的泛素化修饰底物。利用iTRAQ技术研究发现，相较于大豆正常植株，在GmPUB2过量表达转基因植株中共鉴定出110个差异表达蛋白质DEPs，其中35个DEPs下调表达，75个DEPs上调表达；在GmPUB2敲除突变体中共鉴定出125个DEPs，有43个DEPs下调表达，82个DEPs上调表达。