基于提前终止密码子通读机制解析大豆组蛋白H3氨基酸修饰的表观遗传机理研究

Modified amino acid residues in histone proteins are common in all natural eukaryotic organisms, and some of these covalent modifications were proven responsible for enhancing the organisms' tolerances upon environmental stresses. Generally, histone proteins' modifications at the concerned amino residues are presumed to be regulated by the corresponding modifying enzymes after translation. Could the tRNA-amino acid be modified or could specific pre-existing free PTM-amino acid be directly incorporated into the protein sequence during translation via a unique 'editing' system? Up to date, there are two major evidences suggesting an alternative way of protein modification that deviated from the central dogma of molecular biology: I. Besides the known naturally occurring aminoacyl-tRNA which are tailored for the 20 common amino acids, there are nonsense/missense suppressor tRNAs (NMSTs) capable of incorporating diverse non-natural amino acids into proteins. II. NMSTs could suppress/utilize premature termination codon (PTC) during the gene translating process. The above observations prompt us to reconsider the molecular mechanism of protein modifications other than the enzyme induced modification pathways. I have initiated compiling a NMST dataset using bioinformatic approaches, and found that soybean and human are two of these species in this dataset. At the moment, I am verifying the read-through phenomenon of the soybean PTC-H3 gene in human Hep G2 cell line. The preliminary results showed that 15~20% TAA/TAG codons could be suppressed and the TAA could incorporate acetyl-lysine into the translating protein. In this proposed investigation, I will focus on the study of direct incorporation of 'modified' amino acids by PTC in the soybean plants during the translation. This investigation should open a new sight on the epigenetic mechanisms of histone proteins modifications except the enzyme induced pathways.

修饰的组蛋白质氨基酸广泛分布于真核生物中，并能增强生物对环境胁迫的耐受能力。组蛋白氨基酸的修饰通常被认为是在蛋白质翻译过程后由特定的修饰酶引发的。然而，游离于细胞体内那些现成的修饰氨基酸是否可以被直接在蛋白质翻译过程中通过特别的"编码"方式引入合成中的组蛋白序列？已有相关证据：1、细胞内存在组装非天然氨基酸的无义/失义抑制tRNA- - 氨酰tRNA聚合酶的正交对；2、它们能够抑制"提前终止密码子（PTC）"的翻译终止功能，促使翻译中的蛋白质序列延伸并引入修饰的氨基酸。本项目以天然存在此类tRNA的大豆为研究对象。初期试验结果发现，含有PTC的大豆组蛋白H3基因在Hep G2细胞中有15~20%比例的通读率，且发现PTC H3基因中的终止密码子TAA"编码"乙酰化的赖氨酸。本项目将基于TAA/TAG通读机制的研究，从无义/失义突变的基因中探索大豆组蛋白H3上相关氨基酸修饰的表观遗传物基础。

本项目主要围绕大豆提前终止密码子（PTC）通读机制与组蛋白H3修饰的关系展开，目标是从转录过程中引入的组蛋白修饰来解析植物相关逆境响应机制。我们已经完成了以下内容：1、构建了大豆和苜蓿cDNA文库，克隆了典型的2个组蛋白H3的全长cDNA（XM_003589336.1和NM_001248856.1），并在K4、K9、K14、K18、K23、K27、K36、K79位点进行点突变获得含提前终止密码子的DNA片段；2、将上述片段分别构入pEarlyGate 103（含绿色荧光标记）、pDL28-HA载体（含红色荧光标记），共构建表达载体32个；3、将上述表达载体以发根农杆菌k599介导转入大豆毛根分析H3-PTC通读情况。.此外，我们还做了以下研究：1、通过质谱分析一共找到2692个大豆磷酸化蛋白质以及分布在蛋白序列上的5509个磷酸化位点，并初步验证磷酸化的转录因子MYB通过调控的查耳酮代谢进而调节植物耐盐响应；2、建立BP-ANN-QE和GA-BP-ANN模型，分别为植物对特定温区的分布和播种时间提供可靠的预测工具；3、验证接种根瘤菌可以增强大豆耐盐性，并初步验证该功能在很大程度上归功于根瘤菌对查耳酮代谢途径的调控作用。.项目负责人以第一作者或者通讯作者在Molecular & Cellular Proteomics等SCI刊物上的学术论文5篇。培养硕士研究生3人，本科生11人。获得浙江省生命科学竞赛一等奖1项，三等奖1项，浙江省大学生挑战杯三等奖1项。.此外，项目组获得大豆耐盐研究方面授权专利1项。