中国科学院海洋研究所机构知识库

海带长期生活在蓝光占主导的潮下带海水中，蓝光受体在感光之后引发的一系列生理及生化响应，对于其适应潮下带的生境具有重要意义。本研究针对海带的蓝光受体隐花色素、向光素和Aureochrome，结合小RNA、长链非编码RNA（lncRNA）、转录组、降解组和全基因组甲基化分析，探讨蓝光受体相关的非编码RNA调控以及5 mC DNA甲基化修饰，并对参与调控海带孢子体蓝光受体的非编码RNA和DNA甲基化酶功能深入研究。

通过小RNA、lncRNA以及靶基因的关联分析，筛选出海带孢子体中参与调控蓝光受体隐花色素Cryptochrome和Aureochrome的非编码RNAs。海带miRNAs中，novel-m3234-5p与sjCRY-DASH不仅序列互补配对，而且转录本表达呈负相关，推测其可作为负向调控因子。此外6个基因间lncRNA中TCONS_00043396、TCONS_00043393、TCONS_00009907、TCONS_00008371、TCONS_00008286和TCONS_00002718与SjCRY-DASH呈现共表达模式，通过trans作用方式调控sjCRY-DASH。通过与竞争性内源RNA争夺novel-m3234-5p结合位点，sjCRY-DASH可能间接参与DNA损伤修复、蛋白合成和加工以及肌动蛋白转运过程的调控。

与隐花色素广泛分布在植物中不同，Aureochrome仅在不等鞭毛类中发现。海带中有18个miRNAs能够与Aureochrome序列完美配对。其中，已知miRNA中miR8181与aureochrome5转录本丰度呈现负相关，降解组测序分析进一步揭示miR8181对aureochrome5的精确降解位点，证实了miR8181和aureochorme5之间的靶向作用关系。对海带miR8181所有的靶基因功能进行富集分析发现，miR8181参与海带细胞分化和发育过程中的生物学调控。通过不等鞭毛类的三角褐指藻构建miR8181-eGFP过表达株系，使miR8181发生过表达。结果发现，miR8181过表达导致靶基因aureochrome、E3泛素蛋白连接酶、EF2和SET2表达量均显著下调，光捕获能力下调，细胞生长速率显著降低，间接反映出miR8181负向调控aureochrome基因表达，并且干扰细胞正常生长。

通过海带孢子体不同部位DNA甲基化图谱分析，我们发现海带孢子体固着器、柄部和叶片中的5mC DNA总甲基化水平不足0.9 %，且海带基因组中DNA甲基转移酶成员较少，仅包含DNMT2，体外表达证实其融合蛋白分子量接近80 kDa。DNMT2在三角褐指藻中过表达之后其总体DNA甲基化水平提升幅度较小，表明其DNA甲基化催化能力较低。DNA甲基化修饰在海带蓝光受体并非广泛存在，而是仅发生在隐花色素家族成员中的CRY-DASH和向光素（PHOT）中，并且CRY-DASH和PHOT中均存在CG、CHG和CHH三种甲基化修饰类型。通过5-AzaC对海带进行处理，蓝光受体CRY-DASH转录本丰度上调，而PHOT转录本丰度呈现下调趋势，且波动幅度较小，这表明DNA甲基化能够微调CRY-DASH和PHOT的转录。

通过海带表观遗传调控中非编码RNA和5 mC DNA甲基化修饰的研究，阐明表观遗传对海带蓝光受体CRY-DASH、Aureochrome和PHOT调控功能，且不同光受体类型的表观调控模式存在差异，表明潮下带褐藻蓝光受体复杂而精确的调控网络，这对于理解大型褐藻的表观遗传修饰对其适应潮下带环境的作用具有重要理论意义。

The giant brown seaweeds-Saccharina japonica inhabits in the subtidal zone, where the blue light is dominant. Physiological and biochemical responses of blue light receptors triggered by light are important for S. japonica to adapt to the coastal environments. In this study, we integrated the small RNA sequencing, long non-coding RNA (lncRNA) sequencing, transcriptome sequencing, degradome sequencing, and the whole genome methylation sequencing to explore the effect of non-coding RNA regulation and 5 mC DNA methylation modification on blue light receptors (cryptochrome, phototropin and aureochrome) in sporophytes of S. japonica. The functions of related non-coding RNAs and DNA methylase were further investigated to promote the understanding of their regulation in blue light receptors.

According to the integrative analysis of small RNA, lncRNA and their target predicting results, the non-coding RNAs (ncRNA) that targeted to the cryptochrome and aureochrome were screened in sporophytes. Novel-m3234-5p exhibited perfect pairing with sjCRY-DASH and its transcript decreased with the increase of sjCRY-DASH, which may play a negative role in regulating the expression of sjCRY-DASH. Long intergenic non-coding RNAs (lincRNAs), including TCONS_00043396, TCONS_00043393, TCONS_00009907, TCONS_00008371, TCONS_00008286 and TCONS_00002718, exhibited co-expression patterns with sjCRY-DASH. By competing for the binding site of novel-m3234-5p, sjCRY-DASH may indirectly participated in the repair regulation of damaged DNA and protein biosynthesis and processing as well as actin transportation.

Aureochrome is a blue-light receptor specifically found in photosynthetic stramenopiles. In S. japonica, 18 miRNAs perfectly paired with aureochrome. Among these screened miRNAs, miR8181 was negatively correlated with aureochrome5 with high credibility, exhibiting tissue-specific expression in sporophyte of S. japonica. Degradome analysis further revealed the exact cleavage site of miR8181 on aureochrome5, verifying the targeting relationship. Functional analysis of the target genes revealed that miR8181 was involved in the biological regulation of cell differentiation and development in S. japonica. To further verify the function of miR8181, we applied miR8181-eGFP overexpression systems in Phaeodactylum tricornutum, of which was derived from a secondary endosymbiotic event and classified as stramenopile like S. japonica. Overexpression of miR8181 resulted in significant decreases of expression levels of aureochrome, E3 ubiquitin-protein protein ligase, EF2, and SET2, light harvesting and cell growth rates of P. tricornutum, suggesting the negative regulation of miR8181 in cell growth.

To the S. japonica sporophytes of holdfast, stipe and blade, the global DNA methylation levels were less than 0.9% and the content of CHH context is in the dominance. Generally, the members of DNA methylases in S. japonica were less and only included DNMT2. Prokaryotic expression results showed that the molecular weight of the DNMT2 fusion protein is close to 80 kDa. The overexpression of SjDNMT2 in P. tricornutum resulted in the slightly decrease of the total DNA methylation level, which was consistent with weak DNA methyltransferase activity. Among the blue light receptors in S. japonica, 5mC DNA methylation only participated in the modification of CRY-DASH and phototropin (PHOT), which included CG, CHG and CHH three types of DNA methylation. After the treatment of 5-AzaC, the RNA mediated DNA methylation pathway was inhibited, leading to the hypomethylation in sporophyte of S. japonica. Additionally, the transcript abundance of CRY-DASH increased, and the PHOT exhibited slightly decreased tendency, indicating that DNA methylation could fine-regulate the transcript expression of CRY-DASH and PHOT.

In summary, we revealed the epigenetic regulations of ncRNAs and 5 mC DNA methylation on the blue light receptors of CRY-DASH, aureochrome and PHOT in S. japonica. Different light receptors were regulated by specific epigenetic regulation patterns, indicating the complex and precise regulation of blue light receptors in the brown seaweed. These results will enrich our understanding for the function of epigenetic modification in S. japonica in adapting to coastal environments.

摘要……………………………………………………………………….…...…I

Abstract…………………………………………………………………….....…III

第1章 引言 …………………………………………………………..…..…..1

1.1 表观遗传学概述………………………………………………....….…..…1

1.1.1 DNA甲基化修饰………………………………………………..….….1

1.1.2非编码RNA调控……………….…………….…………….….…...…6

1.1.3 染色体重塑 .……………….…………….………….…………….…10

1.1.4 藻类表观遗传调控研究进展 .………………….…………....….…..12

1.2 蓝光受体研究进展………………………………………….……………14

1.2.1 隐花色素…………………………………....…………………..…….15

1.2.2向光素…………………………...…………….…………….……..….17

1.2.3 Aureochrome……………………..………………….…………….…..18

1.3 表观遗传对蓝光受体调控的研究进展…………………………………..19

1.4 海带的分类地位和生活史..………………………………………………20

1.5 研究目的及意义..…………………………………………………………20

1.6 技术路线..............…………………………………………………………21

第2章 非编码RNA参与海带中隐花色素的调节..………….......…..23

2.1 材料与方法………………………………………………………….……..23

2.1.1 实验材料…………………………………....…………………..……..23

2.1.2实验仪器……………………...…………….……………….……..…..24

2.1.3 实验试剂……………………..………………….……………….…....24

2.1.4 实验方法……………………..………………….……………….…....25

2.2 实验结果……………………………………………………...…………....31

2.2.1 海带孢子体miRNA测序、比对和鉴定.………………....…..……...31

2.2.2海带孢子体靶向调控隐花色素的miRNA鉴定..………….……..…..33

2.2.3 海带孢子体中novel-m3234-5p靶向调控CRY-DASH表达......……..33

2.2.4 海带孢子体的lncRNA测序、比对和鉴定……………….…….…...36

2.2.5 LncRNA靶向调控隐花色素基因表达…….……………….……........37

2.2.6 海带孢子体sjCRY-DASH的ceRNAs鉴定…….……….….……..….39

2.3 讨论…………………………………………………………...…………....42

第3章 海带孢子体miR8181对Aureochrome调节功能的研究…..45

3.1 材料与方法………………………………………………………….……..45

3.1.1 实验材料…………………………………....…………………..…..….45

3.1.2实验仪器……………………...…………….……………….……..…...46

3.1.3 实验试剂……………………..………………….………………...…...46

3.1.4 实验方法……………………..………………….……………….….....49

3.2 实验结果……………………………………………………...………….....54

3.2.1 海带孢子体中靶向Aureochrome基因的miRNA鉴定…..…….........54

3.2.2 miR8181靶基因聚类分析….……………….………..…..…..……..….57

3.2.3 降解组测序鉴定miR8181靶基因……..……….…..….……..…....….59

3.2.4 三角褐指藻miR8181过表达功能验证………….....……………...….65

3.3 讨论…………………………………………………………...……...…......69

第4章 海带蓝光受体与DNA甲基化修饰……………………..….....71

4.1 材料与方法………………………………………………………….……...71

4.1.1 实验材料…………………………………....…………………..……....71

4.1.2实验仪器……………………...…………….……………….……..…....72

4.1.3 实验试剂……………………..………………….……………….…......72

4.1.4 实验方法……………………..………………….……………….…......73

4.2 实验结果……………………………………………………...………..........80

4.2.1 海带孢子体DNA甲基化文库构建、测序及分析……..……..…..…..80

4.2.2 海带孢子体蓝光受体DNA甲基化修饰鉴定........……..……..…..…..87

4.2.3 海带孢子体转录组对5-AzaC处理的响应….…...…..…..………..…..88

4.2.4 海带DNMT2功能及特性分析….…..….………………..…..…..…….95

4.3 讨论…………………………………………………………...………........102

第5章 结论…………………………………………………………..…....…105

参考文献…………………………………………………………………….......107

附录……………………………………………………………………….….......123

致谢……………………………………………………………………….......….125

作者简历及攻读学位期间发表的学术论文与研究成果………...…...126