类caspase-3蛋白泛素化在铝诱导花生根尖细胞程序性死亡中的调控作用

This project is to uncover regulation mechanism of caspase-3-like (Ahc-3) protein ubiquitination between different aluminum (Al)-resistant peanut cultivars during Al-induced programmed cell death (PCD) in the root tips of different Al-resistant peanut cultivars. E3 Ubiquitin ligases that interacted with Ahc-3 in Al-resistant and Al-sensitive peanut cultivars, namely ULE-99 and ULE-ZH, respectively, are screened and characterized by using comparative transcriptome analysis and yeast two-hybrid technology. The transcriptional and translational levels of the characterized ULE-99 and ULE-ZH are investigated in corresponding two peanut cultivars. The interaction between ULE-99/ULE-ZH and Ahc-3 and their interacted domains and ubiquitination are detected by using co-Immunoprecipitation, point mutation and in vivo ubiquitination technologies. The recombinant plasmids containing ULE-99 and ULE-ZH, respectively, are constructed and then introduced into wild type tobacco and its mutants TAhc-3, TAhSAG1 and TAhBI-1. The effects on the expression of Ahc-3, SAG1 and AhBI-1 in the constructed transgenic tobacco are observed. PCD is monitored in wild type tobacco and its transgenic plants under Al treatment by DNA ladder, TUNEL and mitochondrial alterations. The conclusion will provide a new sight to understand the regulation mechanism of caspase-3-like protein ubiquitination in PCD and further verify molecular mechanism of Al-resistance in plants.

以铝耐品种和铝敏感品种为材料，以与Ahc-3蛋白（类caspase-3）互作为核心，利用差异转录组学和酵母双杂交等技术，筛选获得不同抗性品种中与Ahc-3蛋白互作的E3泛素连接酶ULE-99和ULE-ZH。利用Nothern blot和West blot检测ULE-99和ULE-ZH在不同铝处理下的转录和翻译水平；利用免疫共沉淀、点突变等试验研究与Ahc-3的相互作用和互作结构域及泛素化水平；构建重组质粒，将基因分别转入烟草野生型及突变体TAhc-3、TAhSAG1、TAhBI-1，检测对Ahc-3、SAG1、AhBI-1等凋亡相关基因表达的影响。通过DNA ladder、TUNLE、线粒体生理等检测野生型和突变型在Al处理前、后细胞程序性死亡（PCD）发生的变化。研究结果对明确不同抗性品种中Ahc-3蛋白泛素化在铝诱导花生根尖PCD中的调控作用与耐铝关系，阐明植物耐铝机制具有重要意义。

铝毒害严重影响酸性土壤中农作物的产量和质量。目前对铝毒害的研究已有较多报告，但植物铝毒害和耐铝机制仍然不清楚。花生是我国的重要油料作物，而对花生铝毒害和耐性机理的研究还较少。通过花生转录组学筛选获得16个与泛素化相关的基因序列，全长克隆，酵母双杂交、CO-IP试验发现其中AhUB4与AhMC1有互作关系，同时通过构建花生Y78酵母库，以AhMC1为诱饵蛋白，通过酵母双杂交筛选到互作蛋白AhMUG，并进一步通过BI-FC及CO-IP试验证实两者存在互作关系，且只通过C端进行互作，并通过酵母三杂交验证了三者之间以AhMC1为桥梁蛋白存在互作关系。同时发现AhUB4定位于细胞膜与细胞质，主要在根、茎部表达，AhMUG主要在花部表达，且都在铝胁迫下在不同的耐铝的花生品种中表达水平差异显著。构建AhUB4、AhMUG和AhMC1表达载体和突变体，分别获得拟南芥转基因植株和突变体植株。通过生理生化、分子和蛋白质水平，发现AhUB4和AhMUG共同促进植物中类caspase基因的转录水平和蛋白表达水平，且在上游发生调控作用，并通过蛋白酶体途径，促进PCD的发生，提高植株的铝敏感性。此外，本项目中增加了花生遗传体系的建立、花生VIGS沉默体系的建立、花生毛状根转化体系的建立，为后续的花生分子育种打下扎实的基础。以上科研成果为阐明花生耐铝分子调控网络提供了重要参考，为通过花生耐铝分子设计育种提供了理论依据、基因资源及实际意义。