白背飞虱基因组解析及其与植物病毒免疫互作研究

Southern black streaked dwarf virus (SRBSDV) is a new species of the Reoviridae family which causes serious diseases of rice. White-backed planthopper (WBPH) has been identified as a higher-efficient transmission vector of SRBSDV. The SRBSDV overcomes both physical and immune barriers of the insect and propagated to a large amount of quantity. The investigation of the interactions among WBPH, SRBSDV and rice has been one of the hot topics for scientists, but advances in the field is largely hindered because of lack of the genome information of WBPH. In this proposal, we intend to construct diverse genomic libraries with different insert sizes for in-depth sequencing by next generation sequencers. These reads will be assembled by computational tools to obtain high-quality genome sequence. Annotation of the de novo assembled genome sequences will be performed, especially for identifying all immune-related genes. Total RNA will be isolated from WBPH infected with/without SRBSDV and will be used for construction of RNA-Seq libraries. Based on the genome sequences of WBPH, all differentially expressed genes will be identified by computational analysis. Combined diverse sources of gene-gene interactions, interaction network of immune genes of WBPH will be built and immune subnetworks containing differential expression genes identified above will be selected for detailed analysis. Unknown genes interacted with known immune genes in the subnetwork will be regarded as candidates of new immune genes. Further experiments will be performed to validate the antiviral function of these genes. Our project will not only provide high-quality genome sequences, but also provide new approaches to understand the complex interactions among the three species.

南方黑条矮缩病毒(SRBSDV)是呼肠孤病毒科一个新种，能引起水稻严重病害。白背飞虱是传播该病毒的专一化介体昆虫，病毒在其体内完成增殖循环后，通过昆虫取食传播给水稻。研究“白背飞虱- SRBSDV -水稻”三者互作是植物病毒领域的热点和难点。由于缺乏白背飞虱基因组信息，目前三者互作的研究进展缓慢。我们拟构建不同片段长度的基因组文库，通过深度测序和组装来获得高质量的白背飞虱基因组序列，进行详细的基因组注释，特别是免疫基因的分析。同时对白背飞虱在感染SRBSDV病毒前后的样本进行转录组建库和测序，利用所获得的基因组信息，鉴定白背飞虱感染SRBSDV病毒后的差异表达基因。我们将充分利用多来源的基因互作数据，构建白背飞虱免疫基因作用网络，研究白背飞虱与SRBSDV互作的免疫子网络，鉴定新的免疫基因并予以实验验证。我们研究不仅提供高质量的基因组序列，而且有助于理解白背飞虱与SRBSDV免疫互作机制。

南方黑条矮缩病毒（SRBSDV）是呼肠孤病毒科斐济病毒属的一个新种，由该病毒引起的南方黑条矮缩病毒病已在我国广大稻区发生，危害严重。SRBSDV传播涉及“白背飞虱-SRBSDV-水稻”三者互作，而目前白背飞虱基因组序列未知，阻碍了白背飞虱传播SRBSDV机制的研究。本课题围绕白背飞虱组学和白背飞虱与SRBSDV互作机制的研究开展了研究工作。 主要完成了: 1) 获得高质量的720Mb的白背飞虱高质量基因组序列, Scaffold的N50大于1M。结合不同发育阶段的RNA-Seq数据鉴定了21,254个蛋白编码基因。2) 获得了白背飞虱的三种共生生物的基因组序列。3) 鉴定了来源于白背飞虱基因组中3,057个位点的3,102个lncRNA转录本和1,706个白背飞虱circRNA,分析了它们在白背飞虱不同发育阶段的表达谱。4）整合了多种类型的基因间相关作用数据，构建了翅膀发育调控网络。使用MCL聚类方法筛选了翅型相关的基因互作网络，并进一步筛选出了45个潜在的翅型调控基因。5）搭建了白背飞虱的组学数据库，整合了基因组序列、基因注释、蛋白功能注释、非编码RNA、基因表达谱、转录因子结合位点、甲基化位点等信息，并且提供了这些数据的检索和下载服务。6）筛选了具有抗病毒效应的白背飞虱miRNA。7）鉴定了白背飞虱的免疫基因集，获得白背飞虱感染SRBSDV诱导的免疫相关基因列表。8）发现了模式昆虫果蝇抗病毒lncRNA(VINR)和沉默抑制子DCV-1A结合，保护VINR不被细胞内RNA酶作用，从而增加VINR在细胞内的含量，并进而激活VINR-Cactin介导的一条非经典免疫通路。我们的研究成果不仅为国内外从事白背飞虱相关工作的研究人员提供了组学数据，同时也有助于理解白背飞虱-SRBSDV之间复杂的免疫互作机制。到2019年底，本课题相关的研究成果已整理发表了8篇SCI论文，包括Cell Host & Microbe、Gigascience、 BMC genomics等业内知名杂志，培养博士研究生2名，硕士研究生2名，本项目达到预期的研究目标。