番茄广谱胁迫蛋白SlUSP介导抗灰霉病的分子机制

SlUSP was cloned from tomato in our previous research, the abiotic tolerance was significantly enhanced in overexpressing SlUSP of tomato, the gene homology is just 58%, when blasted against the GenBank database, and no functional USP genes was reported in plant in the present. The disease resistance was also enhanced significantly in overexpressing SlUSP plants compared to the control when exposed Botrytis cinerea, indicating that SlUSP probably mediated the abiotic and biotic resistance. So we start the study from the signal factors related to the abiotic and biotic resistance, such as Ca2+, ROS and some hormones SA, JA and ET and so on. In order to determine the main signal factors mediated by SlUSP, the transgenic and wild type plants would be treated with hormones or hormone inhibitors, and the expression profile of transgenic and wild type plants would be investigated from the whole genomic level after inoculating Botrytis cinema using RNA-Seq technology, there studies will help to explore which signal and hormone biosynthesis pathway were mediated by SlUSP, and which disease resistance genes were activated finally. Moreover, Yeast one hybrid technology was performed to explore the transcription factors and regulated genes interacted with the promoter elements of SlUSP. This study helps to reveal the new mechanism of plant disease resistance, and to understand the network of abiotic and biotic resistance.

前期我们在番茄中克隆了一个SlUSP基因，在番茄中超量表达后其非生物抗性显著提高，该基因与GenBank数据库中的基因同源性最高仅为58%，目前在植物中未见有功能的USP基因的报道。研究期间发现超量表达SlUSP的植株对灰霉病抗性明显强于对照，表明其能同时介导非生物与生物抗性信号。因此，本研究将从能同时介导非生物与生物抗性的信号因子Ca2+，ROS及激素SA，JA和ET等入手，利用这些信号因子及其抑制剂进行处理，研究由SlUSP介导抗病性提高的主要信号传导途径。运用高通量RNA-Seq技术，在接种灰霉病前后，从全基因组水平对转基因株系和对照的表达谱进行分析，探寻SlUSP介导的信号影响到哪些信号因子或激素的合成，最终激活了哪些抗病相关的基因。进一步，利用酵母单杂交技术，挖掘调控该SlUSP基因的转录因子或调节基因。该研究有助于揭示植物抗病的新机制，理解生物逆境与非生物逆境的网络关系。

我们将野生番茄中SlUSP基因在栽培番茄中超量表达后，发现番茄植株抗灰霉病的能力明显增强，随后我们对其抗病的分子机理进行了研究。我们也构建了干涉SlUSP基因的载体，转化番茄后发现其抗病性降低了。通过构建SlUSP基因启动子与GUS的融合表达载体，转化番茄后，发现ABA、SA和JA均能诱导转SlUSP基因启动子与GUS的植株中GUS的表达。利用酵母双杂交和BIFC技术，我们发现SlUSP可以与MYB96互作。在拟南芥中超量表达MYB96，可以促进SA的合成，从而诱导抗病的PR基因表达，增强植株的抗病性。 超量表达SlUSP后发现与JA信号途径和响应JA的抗病基因表达量得到了提高，如PI-I和PI-II的表达量显著提高了。而与SA相关的基因如NPR1表达量显著下降了。可能超量表达SlUSP的植株中，JA信号途径得到了增强，而JA对灰霉病的抗性起正向作用。由此推断植株的抗病性可能与JA信号的增强有关。另外我们对接种番茄灰霉病后的RNA-seq表达谱分析后，利用VIGS技术我们对20多个差异基因进行了干涉，随后接种灰霉病，结果发现干涉TRV-5830，TRV-0340， TRV-5450这三个基因的病斑面积显著增加，而TRV-4760的病斑面积显著降低。其抗病性变化与活性氧的积累和激素的信号有关。该研究为揭示番茄抗灰霉病的机理奠定了基础。