Kruppel-Like 转录因子调控大连紫海胆性腺脂类物质代谢机制研究

The Dalian purple sea urchin, Strongylocentrotus nudus, is the important cultivation animal due to its economic and health care gonads consisting of high content of lipids. However, research regarding transcript regulation on lipids metabolism is absent yet. In this research, functions of Kruppel-like transcript factor genes upon lipid metabolism will be elucidated. First, KLF 5,7and 15 genes are cloned from gonads of S.nudus at two development stage as materials, and its sequence features such as functional domains, protein-binding motifs and evolution are investigated. Then, the expressional profile with its partner genes, interaction with CtBP and sin3A, co-regulation with C/EBPs in PPARs transcriptional activation are studied by in situ RNA hybridization, transcriptome, BiFC and EMSA methods, respectively. Second, the target genes of KLF-activite PPARs involving in lipid metabolism are identified and characterized by the chromosome immunity co-precipitation-solexa sequencing and bioinformatics tools. Furthermore, the interaction of target genes and PPARs will be valided by EMSA and proteomics approach. Co-relationship analysis of down-stream genes of KLF-activite PPARs with data of species and quantity of lipids in gonad are calculated according Two-Way ANOVA, p<0.01. Third, sea urchin KLF 5,7and 15 gene are introduced into model systems, 3T3-L1 cells and KLF mutation Caenorhabditis elegans, to carry out functional analysis including adipocyte differentiation, lipid metabolism and expressional change of lipid matabolism related genes, thereby the similarity and unique properties of sea urchin KLFs with that of Homo sapiens and C. elegans will be revealed. Collectively, the regulation mechanism and properties of KLFs on lipid metabolism at different development stage in gonad of S.nudus will be presented. The results of this study will not only deepen our knowledge regarding the mechanism of KLFs controlling lipid metabolism at transcript level, but also conducive to breeding and germplasm preservation in sea urchin.

大连紫海胆是重要的经济养殖动物，其性腺脂类物质丰富，具有较好的保健功能。尽管海胆脂类物质合成方面已有较多研究，但是在转录水平研究脂代谢调控机制仍然较为薄弱。本研究以S.nudus海胆性腺为研究对象，利用染色体免疫共沉淀与深度测序技术以及其他多项先进技术，克隆海胆性腺KLF5、7和15基因，分析其序列特征、在生殖细胞和储藏细胞中的表达模式、与共调控因子的结合特点、对PPARs的转录调控功能以及PPARs对脂代谢相关靶基因的转录刺激活性。并结合代谢组学技术分析脂类物质与靶基因表达之间相关性；在此基础上，利用转基因技术，在模式细胞和线虫中表达KLF基因，对其功能特点进行验证，从而在转录和物质代谢水平分析海胆KLF基因对脂代谢的调控机制及作用特点。该研究填补了这方面的研究空白，为进一步全面而深入地研究海胆脂代谢机制提供了重要的理论依据；同时，本研究结果对于指导海胆育种和种质保存也有重要的借鉴价值。

1.研究背景.Kruppel-Like 转录因子(KLF)是调控脂类物质代谢的关键因子之一，但是对海胆其调控机制仍不清楚。本研究克隆了不同KLFs基因，分析KLFs与其他转录因子的互做，初步阐明了KLFs对PUFA代谢的调控机制。.2.主要研究结果.2.1转录组学分析. 转录组学分析结果表明751个基因参与脂代谢。发现了3种类型的fatty acids front-end desaturase (FADSs)和very long fatty acids elongase (ELOVLs)。.2.2 KLF基因的克隆与表达分析. 利用RACE技术克隆了海胆KLF7、KLF13和KLF10基因，同时克隆了CEBPα、CEBPγ和CEBPζ全长序列。KLF7蛋白由291个氨基酸构成，含3个锌指结构，与斑马鱼KLF7进化关系较为接近。基因共表达分析表明，KLF7与C/EBPα、PPARα、PPARγ和PPARδ表达具有明显的相关性，而KLF10与CEBPα (B)、CEBPγ (C)和CEBPζ (D)之间表达相关性模式较为复杂。表明不同KLFs转录调控功能有所区别。KLFs和C/EBPs在二龄和三龄海胆雌雄性腺表达均有差异，发挥重要的作用。.2.3 KLFs的转录调控作用.克隆了上述基因的启动子序列，对启动子序列中包含的转录因子结合DNA的motif进行了分析。同时利用突变技术，确认了核心启动子序列，并在293T细胞中进行了验证。双荧光素酶报告基因实验 结果显示KLF10对CEBPζ(D)的转录激活作用较强，表明它可能是KLF10的主要作用靶点之一。此外，过表达分析表达KLF10和KLF8均抑制C/EBPα、C/EBPγ和C/EBPζ转录，然而KLF13抑制C/EBPγ，增加C/EBPα和C/EBPζ的转录活性。.2.4 海胆FADS和ELOVL基因克隆表达模式分析.克隆了2个FADS和1个ELOVL基因的全长序列，它们性腺组织中高表达。过表达分析表明SnFAD1和SnFAD2具有去饱和酶活性，SnEl能延长18碳和20碳底物。这些基因表达水平与KLFs之间存在相关性。.2.5 KLFs相关lncRNA的挖掘.分析了性腺中的lncRNA和miRNAs，靶基因和ceRNA分析的初步分析表明存在靶向各KLFs的miRNA基因以及ceRNA调控机制。