水稻蛋白异天冬氨酸甲基转移酶OsPIMT1对稻谷耐储性的作用机制研究

Deterioration and loss of viability of rice grains during storage make a great impact on national food security and rice breeding. With the rapid development of molecular biology technology, the use of modern genetic engineering combined with field trials is a main trend on understanding the mechanism of rice storability and breeding rice varieties with high storability. The protein L-isoaspartyl Methyltransferase (PIMT), an important repair enzyme for protein damage，is probably involved in maintaining seed vigor. Nevertheless, PIMT has not been reported in rice yet. Through our early work, it was confirmed that Oryza sativa Protein L-isoaspartate Methyltransferase OsPIMT1 plays a major role in seed storability, but its mechanism remains unclear. Therefore, this project combines two methods, Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) and PIMT-based protein on-blot methylation analysis, to identify the relation between two types of proteins during artificial ageing of seeds：1) isoaspartyl-containing proteins repaired by OsPIMT1, as well as 2) differentially expressed proteins related with seed ageing and vigor loss, using the stable OsPIMT1 transgenic lines and wild type Nipponbare as materials. The mechanism of OsPIMT1 in seed storability is expected to be clarified, which could provide a theoretical basis for the breeding of rice varieties with high storability.

稻米储藏过程中的陈化变质以及种子活力下降的问题，影响国家粮食安全和水稻育种工作。随着分子生物学技术的快速发展，利用现代基因工程结合田间试验来了解耐储藏机理并选育优良的品种是水稻种子耐储性研究的重要方向。蛋白异天冬氨酸甲基转移酶作为植物中重要的蛋白损伤修复酶,可能在维持种子活力具有突出作用。此类酶在水稻中的功能尚未研究过。申请者通过前期的工作，确定了蛋白异天冬氨酸甲基转移酶OsPIMT1的表达提高了水稻耐储性，但其作用机理仍不明确。本项目拟利用稳定表达的OsPIMT1转基因株系与野生型日本晴作为材料，通过同位素标记相对和绝对定量分析和基于PIMT修复的印迹甲基化分析相结合，找到种子人工老化过程中两类蛋白质之间的关系：1. 被OsPIMT1修复的上游异构蛋白；2.种子老化与失活相关的下游差异蛋白。可望解析OsPIMT1对于种子耐储性的作用机制，为选育耐储藏水稻品种提供理论依据。

稻米储藏过程中的陈化变质以及种子活力下降的问题，影响国家粮食安全和水稻育种工作。随着分子生物学技术的快速发展，利用现代基因工程结合田间试验来了解耐储藏机理并选育优良的品种是水稻种子耐储性研究的重要方向。蛋白异天冬氨酸甲基转移酶PIMT作为植物中重要的蛋白损伤修复酶,可能在维持种子活力具有突出作用。此类酶在水稻中的功能尚未研究过。申请者通过分析过表达以及RNAi种子老化过程中种胚异构天冬氨酸蛋白的积累，证实了OsPIMT1介导的种子寿命提高很可能是由于修复了种胚中过量累积的含isoAsp的异构蛋白。随后，通过iTRAQ蛋白组学分析获得了65个老化相关蛋白，并进行了GO功能富集和KEGG代谢途径富集分析，将抗氧化系统和脂类代谢作为重要候选途径，同时利用印迹甲基化分析RNAi材料种胚中的异构蛋白，获得了15个含异构天冬氨酸的蛋白，其分别与脂类运输和储藏、抗氧化、能量代谢等相关。结合二者的分析结果，选取了脂类运输储藏蛋白Olesin-18kd以及Olesin-16kd、酰基辅酶A结合蛋白、脂转运蛋白以及过氧化物酶2作为候选基因。通过T-DNA插入突变体库以及Crispr/Cas9技术分别获得了这5个基因的缺失突变体，为后续进一步明确OsPIMT1介导的水稻种胚蛋白组修复机制奠定基础。