基于表观遗传学分析探讨珍珠贝的移植免疫调控机制

Transplantation operation was the most crucial process during pearl production. After the transplantation operation, the immune rejection response would be induced by the transplanted mantle tissue and nucleus, even so, pearl sac was finally formed and secreted the nacre which was responsible for pearl formation. The regulation mechanism underlying rejection to coexist between the donor and the receptor was interested by the researchers. Though many immune related-genes have been obtained and functionally studied, however, the modulation mechanisms of the transplantation immunity in the pearl oyster were not clear until now. Pinctada martensii was the main species cultured for pearl production in China. Our previous research showed the genes expression of miRNA process and DNA methylation related enzymes were changed significantly at different times after the transplantation operation in the hemocyte transcriptomes of pearl oyster, which indicated that epigenetic were involved in the modulation of transplantation immunity after the transplantation operation. Based on the finished analysis of the genome and transcriptomes of Pinctada martensii, the aim of this study was to detect the dynamic changes of epigenetic regulators, including microRNA (miRNA) and DNA methylation. Combined the analysis of genome and transcriptomes, we could obtain the crucial epigenetic regulators and the related target genes. Further, we could verify and functionally study the epigenetic regulators and the target genes by various molecular technologies, and finally elucidate the function and mechanism of epigenetic regulators in the transplantation immunity during pearl production in pearl oyster. Meanwhile, this study would provide the theoretical basis for further elucidating the mechanism underlying immunity in invertebrate.

植核手术是珍珠生产过程中的核心环节，植核后异体移植的外套膜组织和珠核会引发受体贝的免疫排异，但最终能形成珍珠囊并分泌珍珠质形成珍珠，这种供体和受体之间从排异到共存的分子机制一直备受关注。虽然，目前已获得数十种功能基因，但珍珠贝如何协调控制这些基因的表达，并发挥其相应功能的机制尚未明了。课题组前期的研究发现我国海水珍珠培育的主要物种马氏珠母贝miRNA加工和DNA甲基化等表观遗传调控相关酶的基因表达在植核后发生了较大变化，说明珍珠贝植核后的免疫反应受到表观遗传的调控。本项目拟在已完成马氏珠母贝基因组及转录组分析的基础上，系统研究miRNA和DNA甲基化在植核后的动态变化，结合基因组和转录组分析获取关键的miRNA和DNA甲基化，以及miRNA和DNA甲基化之间的调控关系，并采用多种分子生物学技术进行验证和功能分析，以期揭示珍珠贝的移植免疫调控机制，为无脊椎动物免疫机制研究提供理论参考。

深入解析植核后供受体间从排异到共存的移植免疫机制对培育优质珍珠至关重要。本研究通过分析植核后不同时间血细胞miRNA转录组发现，马氏珠母贝血细胞含有186个miRNAs，其中包含99个已知miRNAs，87个novel miRNAs，并重点分析了novel-63和miR-1985在移植免疫中的作用和机制。结果表明，过表达novel-63可以抑制血清多种免疫酶活性，而过表达miR-1985促进血清免疫酶的活性；转录组分析表明，体内过表达novel-63导致Notch等信号通路发生改变；过表达miR-1985导致ECM-receptor interaction等信号通路发生改变；实验证明novel-63的靶点有GDP-FucTP、CysLTR2和RLR；miR-1985的靶点有GATA-3、RIG-I like和IFI44L。同时，我们分析了nAChR、Myd88、IL-17、HSP40等移植免疫基因的进化历程，并验证nAChR-1与miR-873、nAChR-3与miR-4577、nAChR-4与miR103a、nAChR-5与miR-6753、Myd88与MiR-4047之间的靶向关系。WGBS分析表明，马氏珠母贝血细胞DNA中2.6%的C碱基被甲基化修饰，并且CDS的甲基化程度最高，同时发现基因的表达水平与甲基化修饰呈正相关。植核后6h的差异甲基化区域（DMR）最多，其相关基因主要富集在细胞增殖和免疫响应相关通路。有的基因（如NF-kb、IKB、IAP6）在植核后甲基化程度增加，有的基因（如nAChR、TLR4和IAP3）甲基化降低；转录因子分析表明，DNA甲基化修饰可能通过改变转录因子（如SP1、C-Jun和CREB）的结合来调控目的基因的表达。植核育珠实验表明，抑制细胞增殖相关基因可以提高植核贝的留核率。同时，我们克隆获得三个DNA甲基转移酶的基因全长，并分析了它们的组织表达模式。靶向分析表明，DNMT1a的调控miRNA有miR-184-3p，DNMT3的调控miRNA有miR-750_1。过表达miR-184-3p可以有效促进血清中的多种免疫酶的活性，PAMP刺激和植核手术都可以诱导miR-184-3p的表达；并且，miR-184-3p和DNMT1a的表达受DNA甲基化的调控。本研究为有效调控珍珠贝植核后的移植免疫提供理论基础。