组蛋白乙酰转移酶p300基因OsHAC701在水稻低温应答中的功能研究

Protein post-translational modifications play a major role in various cellular processes, such as transcriptional regulation, protein interaction, and modulating enzymatic activity. A number of different types of PTMs have been reported, including phosphorylation, methylation, ubiquitinoylation, acetylation, and crotonylation. Lysine acetylation (Kac) have been widely studied in prokaryotes and eukaryotes as a particularly dynamic and reversible type of PTM, while lysine crotonylation (Kcr) is a newly discovered PTM that exists in mammals and dicots. Histone acetylation is an important epigenetic regulation in higher plants adapting to different environmental conditions. Histone acetyltransferases may activate transcription of stress response genes, thereby regulating plant metabolic and physiological processes including cold, salt and drought stress responses. Similar to acetylation, histone crotonylation catalyzed by the histone acetyltransferase p300 has been proved crucial for directly stimulating transcription, controlling gene expression and epigenetic regulation. However, the research on them is quite limited in plants. .In the previous work, we found that histone crotonylation are extensively distributed in rice, and plays an active role in regulating gene transcription in the rice genome, as well as histone acetylation. In order to explore the epigenetic regulation mechanism of these two modifications, we selected the histone acetyltransferase p300 as research object. By using CRISPR/cas9 gene editing technology, the p300 family gene OsHAC701 was successfully knocked out in rice. Compared to the wild type, phenotypic observation of Oshac701 mutant showed dwarf plant type, small seed size and low setting percentage. And western blot results indicated the levels of both histone acetylation and crotonylation were significantly reduced in Oshac701 mutant. Previous reports showed that histone Kac is involved in regulating plant responses to salt, cold, and other abiotic stresses. After treated with cold (4℃) for 10 h, western blot analysis revealed that acetylation of both H3K27 and H3K36 and crotonylation of H3K14 are elevated significantly as compared with the control group. In addition, the expression level of OsDREB1A, a cold stress response gene, significantly raised, while histone crotonylation was significantly enriched in the gene promoter regions. The above results suggest that histone acetylation and crotonylation may participate in the epigenetic regulation of cold stress responses in rice. .The project is proposed on the basis of the comprehensive utilization of molecular genetics, proteomics, epigenomics and interdisciplinary research means, to understand the genetic networks of histone acetylation and crotonylation of the regulation on rice genome, and to further discuss the epigenetic regulation mechanism of histone acetyltransferase p300 in plant growth and cold stress response.

组蛋白乙酰化和巴豆酰化修饰在生物生长发育和逆境胁迫等生命过程中起着重要作用，这两种修饰均可以通过组蛋白乙酰转移酶p300进行催化。然而关于它们的研究在植物很少有见报道。在前期工作中，我们发现水稻中广泛存在组蛋白乙酰化和巴豆酰化修饰，并且与基因的表达正相关。利用CRISPR-Cas9技术已成功敲除水稻中p300家族基因OsHAC701，突变体表现出矮小、小粒、结实率低，同时组蛋白乙酰化和巴豆酰化修饰水平明显下调。另外在低温逆境条件下，组蛋白乙酰化和巴豆酰化修饰水平明显上调，低温应答基因OsDREB1A表达显著上调，并且该基因启动子区域巴豆酰化修饰明显富集。本项目拟在此基础上综合利用分子遗传学、表观基因学和蛋白组学等多学科交叉研究手段，重点解析组蛋白乙酰化和巴豆酰化在水稻低温应答中的遗传网络，探讨p300基因OsHAC701在植物生长发育和调控水稻低温应答的表观调控机理。

组蛋白酰化修饰在植物生长发育和响应逆境胁迫过程中发挥重要作用。其主要由组蛋白乙酰转移酶和组蛋白去乙酰化酶共同催化调控，是一种动态可逆的修饰状态。通过本项目的实施，创制了水稻中组蛋白乙酰转移酶p300家族相关基因OsHACs、GNAT家族基因GCN5和组蛋白去乙酰化酶基因HDAC706相关遗传材料，研究表明相关基因突变后影响到水稻组蛋白乙酰化修饰水平，进而影响到水稻植株的正常生长发育，不同基因对植株表型影响程度不同。进一步通过WB、qRT-PCR、ChIP-PCR、RNA-seq、ChIP-seq等研究技术明确了水稻组蛋白酰化修饰响应多种逆境胁迫。在盐胁迫下，GCN5会抑制Na离子转运基因表达；OsHDA706通过H4K5和H4K8去乙酰化调节盐胁迫响应基因OsPP2C49的表达。在低温逆境下，组蛋白H3K14ac、H3K14cr的修饰水平明显上调，而H3K18cr修饰水平明显下调。乙酰化修饰水平发生变化的基因数多于巴豆酰化修饰，其中低温逆境响应基因DREB1A的启动子与基因体上H3K14ac/cr的修饰水平明显上升；H3K18cr通过调控OsDREB1A、OsEATB、OsAP2-39、OsNAC9等转录因子的表达来参与水稻低温胁迫的响应过程。通过相关性分析发现，在低温胁迫下H3K14ac/cr修饰水平上调且差异表达上调的基因都富集在胁迫响应进程上。相关研究结果为解析组蛋白乙酰化和巴豆酰化调控植物响应盐和低温胁迫的表观遗传机制提供遗传材料和理论依据。相关结果发表标注SCI论文5篇，中文权威1篇；培养博士后2名，博、硕士生9名。项目主持人龚志云获2022年江苏省“十佳研究生导师团队”提名奖。

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