【作者】 温荣；

【导师】 李富花；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2014， 博士

【摘要】 对虾是重要的水产养殖种类之一，由于各类病害的爆发，特别是白斑综合症的威胁，对虾养殖产业经受了巨大的损失。对虾抵御病原侵袭的方式是启动先天免疫应答系统，特别是通过信号通路的启动完成对病原的识别和清除。已有研究表明，对虾先天免疫系统中两条重要的信号通路：Toll通路和JAK/STAT通路可能参与了对虾应答白斑综合症病毒的过程。然而，有关对虾Toll通路的关键接头分子MyD88的功能尚未报道，JAK/STAT通路的转录因子STAT在对虾感染WSSV过程中的功能尚不清楚。基于上述问题，本文对对虾的MyD88和STAT在WSSV侵染过程中的作用进行了研究，研究进展如下：1.从中国明对虾中克隆获得了MyD88的同源基因---FcMyD88，其cDNA全长1434bp，编码477个氨基酸。推导的氨基酸序列中包含死亡结构域（deathdomain，DD）和TIR结构域（typical TLR and IL-1R-related，TIR），在其C端有一段约100个氨基酸组成的C端延伸区域（C-terminal extension，CTE）。定量PCR和免疫印迹分析发现，FcMyD88mRNA在藤黄微球菌（Micrococcus luteus）和鳗弧菌（Vibrio anguillarum）刺激后的24小时内均上调表达；FcMyD88蛋白在V. anguillarum刺激的12小时上调表达，而在M. luteus刺激后无明显变化；FcMyD88mRNA虽然在WSSV刺激后表达量相对稳定，但是FcMyD88蛋白在WSSV刺激后明显上调表达；用WSSV感染MyD88沉默后的对虾，由WSSV感染引起的对虾死亡率显著降低。以上结果表明MyD88依赖的信号通路参与了对虾抵御细菌和WSSV感染过程，MyD88可能有利于WSSV的感染。2.利用dsRNA干扰技术沉默对虾STAT基因后再感染WSSV，观察WSSV在对虾中的拷贝数和由WSSV感染引起的对虾死亡率，结果表明，在STAT基因沉默后的对虾中，WSSV拷贝数维持在102-103copies/ng DNA，显著少于对照组，并且其死亡率也显著低于对照组。为进一步了解STAT基因的功能，又利用培养的螯虾造血细胞进行了研究，利用STAT3特异抑制剂（S3I-201）抑制鳌虾造血细胞的STAT活性后再用WSSV感染细胞，其细胞存活率显著高于未经抑制剂处理的对照组，说明STAT在WSSV感染对虾的过程中起着重要作用。为了解在STAT干扰后的对虾中STAT通路可能调控的目的基因以及RNAi调控通路的相关基因的表达，利用实时定量PCR技术对STAT干扰后的对虾中相关基因的表达进行了分析，结果表明，与注射EGFP双链RNA的对照组相比，在STAT被干扰的对虾中再感染WSSV后，GILT, Dicer2和AGO2基因的表达明显上调，而AGO1和Dicer1基因的表达没有发生明显变化，说明STAT被干扰后的对虾可能通过增强siRNA通路达到抗WSSV感染的目的。3.利用高通量的RNA-seq技术筛选STAT被干扰的对虾中差异表达的基因，结果表明，STAT干扰引起了参与能量代谢、糖代谢、外骨骼组成、转录及免疫等生命过程的基因表达的变化。其中，与能量代谢相关酶类，如腺苷酸环化酶、细胞色素c氧化酶I亚基；外骨骼组成相关蛋白，如Culticle、DD5、DD9A；转录相关分子，如转录起始因子TFIID和RNA解旋酶等基因的表达均下调。而与糖代谢相关分子、免疫相关分子等的表达既有上调也有下调，说明STAT干扰改变了对虾体内许多基因的转录表达，这些基因的表达变化可能不利于WSSV在对虾体内的复制。本研究筛选的对虾STAT潜在调控的基因信息，为进一步分析JAK/STAT对虾免疫中的功能奠定了重要基础。更多还原

【Abstract】 Shrimp is the one of the major species in aquaculture. Disease is always aproblem which hinders the healthy development of shrimp aquaculture. Both virusand bacteria are dangerous pathogens to shrimp aquaculture. Shrimp defense againstpathogens mainly by initiating the innate immunue signal pathways. Toll andJAK/STAT signaling pathways play key roles in the antiviral immunity of mammals,fish and insect. However, limited knowledge is known about the function of MyD88and STAT in shrimp, which are regarded as an key component of Toll or JAK/STATsignaling pathway respectively, during WSSV infection. The present thesis mainlyfocused on the function of MyD88and STAT in Penaeid shrimp. The main progressesare as follows:1. The full length cDNA of a MyD88homologue (FcMyD88) was cloned frompenaeid shrimp Fenneropenaeus chinensis. The ORF of FcMyD88consisted of1434bp encoding477amino acids, which contains a death domain (DD), a typical TLRand IL-1R-related (TIR) domain and a C-terminal extension (CTE). Homologyanalysis revealed that the deduced amino acid sequence of FcMyD88shared morethan47%similarities with a variety of previously reported MyD88s. Thetime-dependent expression patterns of FcMyD88in cephalothoraxes of shrimpsinjected by heat killed Vibrio anguillarum (G-), Micrococcus luteus (G+) and livewhite syndrome spot virus (WSSV) were analyzed at transcription and protein levelby qPCR and Western Blot, respectively. The expression of FcMyD88mRNA wassignificantly up-regulated during24h after injection with both V. anguillarum and M.luteus. The expression level of FcMyD88protein was up-regulated at12hour postinjection (hpi) of V. anguillarum, while it didn’t change after M. lysodeikticu injectionfor12h. After WSSV injection, the expression level of FcMyD88mRNA wasrelatively constant, while the FcMyD88protein level was significantly up-regulated at12and24hpi. Further, dsRNA interfering technique was used to silence the expression of MyD88in Litopenaeus vannamei, and the mortality of shrimp wasreduced significantly compared to their controls during WSSV infection. These resultssuggested that the MyD88-dependent signaling pathway could be involved in thedefense of shrimp both to bacteria and WSSV infection, and shrimp MyD88mightbenefit to WSSV infection.2. In order to understand the function of JAK/STAT signaling pathway in theantiviral immunity of shrimp, dsRNA interfering technique was used to silence theexpression of STAT gene in Litopenaeus vannamei, and the mortality of shrimp wasdetected after WSSV infection. The WSSV copy number in STAT silenced shrimp was102-103copies/ng DNA which was much lower than those in the control and themortality in STAT silenced shrimp caused by WSSV infection reduced verysignificantly compared to their controls. The function of STAT was verified in vitrocultured cells of hematopoietic tissue of crayfish Cherax quadricarinatus by addingspecific inhibitor of STAT3(S3I-201), and the cultured cells treated with S3I-201showed much less WSSV copy number than their controls, which further suggestedthat STAT might be helpful for the replication of WSSV. Furthermore, the expressionprofile of some potential target genes regulated by STAT or genes related to RNAinterfering pathway were detected in STAT silenced shrimp during WSSV infection.Dicer2and AGO2were up-regulated in STAT silenced shrimp, which implied thatsilencing of STAT might enhance the expression of siRNA pathway related genes toresist the WSSV infection.3. The differentially expressed genes (DEGs) in STAT silenced shrimp wasscreened by high throughput RNA-Seq. The data showed that silencing of STATcaused transcription variations of genes related to energy metabolism, glucosemetabolism, composition of the exoskeleton, immune response and transcription.Among the DEGs, genes encoding enzymes related to energy metabolism such asadenylate cyclase and cytochrome c oxidase subunit I, genes encoding proteins relatedto exoskeleton compositions, such as cuticle protein, DD5and DD9A, and genesencoding transcription related factors such as ATP-dependent RNA helicase A,,transcription initiation factor TFIID etc. showed down-regulation. Genes related to glucose metabolism and immunity showed up or down regulations, which suggestedthat interference of STAT changed the expression of a lot of genes. The variations ofthese genes’ expression might not be benefical to the replication of WSSV in shrimp.,These information about the potential genes regualted by STAT will help us tounderstand the function of JAK/STAT pathway in the immunity of shrimp.更多还原