诱导玉米杂交种劣变的靶向mRNA氧化研究

Deterioration is one of the important theoretical problems unsolved in the field of seed biology so far. Many studies have documented that the oxidative damage to proteins resulted from the production and accumulation of reactive oxygen species (ROS) is an important cause that leads to seed deterioration. However,whether mRNAs oxidation occuring in embryo is related to the seed deterioration or not still remains unclear to our knowledge. To study the relashionship between the mRNAs species oxidation in embryo and seed deterioration, we will use a hybrid maize cultivar, zhengdan 958, as the experiment model，which have different degrees deterioration caused by natural and artifical seed aging treatment. Three aspects will be investigated as follows: (1)Digital Gene Expression Profiling(DGE) technique will be performed to investigat the changes in mRNAs in different degrees deterioration embryos.(2)The OxiSelect Oxidative RNA Damage ELISA kit,the rabbit reticulocytes lysate system kit (Promega) and the 2-D electrophoresis protocol will be used to assess whether oxidation of mRNAs can be induced and their oxiditive degree.(3)Oxidized RNA immunoprecipitation and microarray hybridization on GeneChip Array will be done to examine whether mRNA oxidation is random or targeted, and these highly targeted mRNAs will be verifed by qRT-PCR. Comparion of the differenc of mRNAs oxidation in various aging embryo may help us to discover the regularity of mRNAs oxidation and understand the relationship between the targeted mRNAs oxidation and seed deterioration. We also want to construct a model about maize seed deterioration based on targeted mRNAs oxidation in embryo. This project will provide scientific base for uncovering the molecular mechanism of seed deterioration.

劣变是种子生物学领域迄今未解的重大理论问题之一。多数研究普遍认为：由活性氧产生和积累所造成的种胚功能蛋白氧化损伤是导致种子劣变的重要原因。然而，种胚中的mRNAs氧化与种子劣变的关系如何未见详细报道。本研究拟以自然老化和人工老化处理获得的具有不同劣变程度的玉米杂交种郑单958为材料：①用"数字表达谱技术"研究不同劣变种胚mRNAs差异；②用8-OHG的单抗免疫技术、体外翻译实验和双向电泳技术研究不同劣变种胚mRNAs氧化损伤程度；③用免疫共沉淀和芯片杂交相结合技术研究不同劣变种胚 mRNAs氧化的靶向性，并对高度靶向性的mRNAs进行QRT-PCR验证；研究自然老化和人工老化处理过程中种胚mRNAs氧化损伤规律，阐明种胚mRNAs氧化与种子劣变的关系，进而构建基于种胚mRNAs氧化损伤的玉米种子劣变模型，为揭示种子劣变的分子机理提供科学依据。

种子劣变是种子生物学领域迄今未解的重大理论问题。本项目研究表明，人工老化过程中种子含水量表现升高趋势，种胚含水量高于胚乳；TTC染色的结果显示：劣变3和5 d的种子，其种胚染色较浅，说明种子劣变3 d后种胚生活力明显降低。发芽试验表明，劣变1 d的种子发芽率几乎没有明显变化，随着处理时间的延长，发芽率出现不同程度的降低，劣变处理3的种子发芽率下降幅度较大，说明劣变处理3 d是种子活力下降的拐点。利用“数字基因表达谱技术”了研究劣变种胚mRNAs表达谱差异，结果表明：人工劣变处理（CDT）处理后玉米种胚差异表达基因有4713个。其中上调表达2874个，下调表达1839个。这些基因涉及细胞组分、分子功能和生物学过程方面,其编码的蛋白主要分布在细胞器和细胞膜上，参与能量代谢、信号转导、刺激响应和衰老死亡等过程，具有结合、催化和抗氧化等功能。其中，能被KEGG数据库注释的有2470个，参与了288条代谢通路,其中有16条通路存在着显著性富集。这些通路中，参与能量代谢的基因共有113个，分别参与糖酵解/糖异生过程(59个)、磷酸戊糖途径(31个)和丙酮酸代谢过程(50个)；其中,调节糖酵解/糖异生过程的烯醇化酶基因、3-磷酸甘油醛脱氢酶基因等、丙酮酸代谢中的丙酮酸激酶基因等和磷酸戊糖途径中的α-L-岩藻糖苷酶基因等上调幅度最大。还发现,调节NADH代谢相关的基因有25个(9个上调，16个下调); NADPH代谢的有10个。这些基因可能导致ROS等自由基的产生和积累，从而加速玉米种胚细胞的衰老及死亡，最终表现种子劣变和活力丧失。因此，水分是种子劣变的关键因素，差异表达基因可能扮演重要角色。本研究可为进一步阐明种子劣变分子机理提供科学依据。. 项目执行期间，分别在《中国农业科学》、《植物生理学报》发表研究论文2篇；培养研究生6名。