银鲫不同雌核生殖克隆系对鲫疱疹病毒抗性差异的调控机制研究

Gibel carp is an important aquaculture species in China, and Carassius auratus herpesvirus (CaHV) has hampered aquaculture development. In previous study, we performed herpesvirus challenge experiments in three gynogenetic clones of gibel carp, including leading variety clone A+, candidate variety clone F and wild clone H, and found that the three clones showed distinct resistance to herpesvirus. Based on the comparative transcriptome profiling between diseased individuals and control individuals in clone A+, F and H, we will firstly identify all gene copies of important interferon system genes from the draft genome of gibel carp clone F and reveal the roles of their diversification to adaptive evolution of gibel carp through comparative genomic analysis. Then, we will identify the important IFN genes related to CaHV resistance difference by analyzing the differential expression changes of IFN system genes among the three gynogenetic clones of gibel carp during different CaHV infected stages, and reveal their function and signal pathway. Furthermore, the differential regulative mechanism to CaHV resistance among three gynogenetic clones of gibel carp will be revealed through promotor analysis. Finally, we will try to apply the techniques of BAC transfer and genome editing to breed novel strain of gibel carp with higher resistance to CaHV. This study will provide a typical example for adaptive evolution of duplicated genes of teleost polyploid, and will benefit to the gibel carp disease-resistance molecular breeding.

鲫疱疹病毒CaHV已经严重制约了异育银鲫的可持续养殖。在分析CaHV易感克隆系A+、中等抗性克隆系F以及高抗克隆系H在感染CaHV前后头肾转录组基础上，本项目拟从银鲫F系全基因组序列中鉴定主要IFN系统基因的所有基因拷贝，通过比较基因组学揭示多倍体银鲫IFN系统基因的多样性及其在进化上对银鲫适应性的作用；分析抑制病毒复制的viperin等主要IFN系统基因的不同基因拷贝在3个克隆系感染CaHV后免疫器官中的表达变化，筛选与抗CaHV紧密相关的关键基因，揭示抗病毒功能和信号通路；通过启动子分析筛选与银鲫CaHV抗性相关的关键位点，揭示银鲫不同克隆系间IFN系统调控机制差异；最后拟通过BAC转基因技术和基因组编辑技术探索培育银鲫抗病新品种的技术途径。本项目的研究结果不仅可以为揭示鱼类基因组加倍后重复基因的适应性进化机制提供一个典型范例，也可为异育银鲫分子设计育种奠定技术平台。

鲫疱疹病毒CaHV已经严重制约了异育银鲫的可持续养殖。本项目在分析CaHV易感克隆系A+、中等抗性克隆系F以及高抗克隆系H在感染CaHV前后头肾转录组基础上，进一步对它们在感染CaHV后存活个体间的转录组分析，三个雌核生殖克隆系的免疫和循环系统相关基因的表达变化既存在共性，但又有所差异，而先天免疫和适应性免疫之间的差异调节机制可能是导致3个雌核发育克隆系对CaHV抗性差异的关键原因。进一步鉴定筛选了viperin、cxcr4、dicp、ptpn6等与银鲫不同雌核生殖克隆系CaHV抗性差异相关的关键基因，揭示了它们的抗病毒功能、信号通路以及调控机制。发现CgViperin-A的缺失主要影响免疫相关途径，削弱了银鲫对CaHV的抗性，而CgVperin-B的缺失则则影响自噬相关途径，对CaHV抗性没有影响。当CgViperin-A和CgVperin-B同时被破坏时，导致银鲫抗CaHV能力完全丧失。体外泛素化实验中发现CgViperin-A和CgViperin-B均可通过减少K63连接的多聚泛素化促进一个宿主IFN负调控因子CaHV ORF46R的蛋白酶体降解，而CgViperin-B还可以通过自噬体途径介导ORF46R的降解。发现银鲫DICP为抑制型免疫受体，通过胞外免疫球蛋白结构域特异性识别脂质A配体，并利用胞内的免疫受体酪氨酸抑制基序招募SHP-1和SHP-2等信号分子，传递抑制性信号，最终抑制干扰素以及干扰素诱导基因的表达。发现cxcr4/cxcl12通过JAK/STAT和MAPK通路增强银鲫抗病毒反应。在六倍体银鲫中建立了CRISPR/Cas9基因编辑技术，以免疫负调控因子SHP（基因为ptpn6）为靶基因，获得分别编辑1个和2个ptpn6等位基因的F2代银鲫突变系，其中编辑一个等位基因的ptpn6+/+/-抗鲫出血病能力比异育银鲫“中科3号”提高25%。本项目的研究结果不仅可以为揭示鱼类基因组加倍后重复基因的歧化进化命运提供典型范例，也可为异育银鲫分子设计育种奠定技术平台。