【作者】 许宝红；

【导师】 肖调义； 章怀云；

【作者基本信息】 湖南农业大学 ， 水生生物学， 2012， 博士

【摘要】 草鱼出血病长期制约草鱼养殖的健康可持续发展。目前,在草鱼出血病病原、病毒生物学、疫苗研制、免疫相关基因克隆以及转基因抗病草鱼研究方面取得了一系列重要科研成就,但一直未能在遗传育种角度取得草鱼抗病育种的突破性进展。本文采用转录组技术筛选与抗草鱼出血病病毒紧密相关的信号因子,试图为后续分子标记辅助育种或转基因育种方法解决草鱼出血病问题提供基础。本研究选取1龄草鱼种,对感染草鱼出血病毒前后的草鱼脾脏进行转录组测序；分别取注射草鱼出血病毒后Oh、24h、48h、72h、96h、138h6个时间点,胸腺、鳃、肝脏、脾脏、头肾5个组织,通过Real-time PCR方法检测筛选出的差异表达因子的时序表达,进一步验证信号因子与草鱼出血病的相关性；实验获得如下创新性研究结果：1、感染草鱼出血病毒前后草鱼脾脏转录组数据库共测2G+2G的数据量,测得4G转录组数据量,共得到109077条unigenes,其中包含108092条差异表达的unigenes,其中差异倍数在2倍以上的unigene数为32621条。其中上调20825条,下调11796条。2060条unigene核苷酸在KEGG数据库中未能预测编码蛋白质,可作为考虑新基因寻找点。2、筛选出2条与草鱼出血病紧密联系的信号通路共获得显著富集的免疫相关通路32条,其中差异最显著的为凝血-补体通路、造血细胞谱系通路、细胞吞噬体通路和细胞-细胞受体互作通路4条,差异表达显著的unigene多集合在凝血-补体通路和细胞-细胞受体互作通路这2条通路中。3、筛选出9个与草鱼出血病紧密相关的信号因子并探明其在草鱼攻毒草鱼出血病毒后的周期性表达规律荧光定量检测数据结合草鱼出血病症状,确定C3、C5、XCR1、F7、F2、PLG、 NGFR、CCR4、XCR1、X9个因子可能跟草鱼出血病毒诱导紧密有关的信号因子。4、首次克隆了草鱼补体因子C5-1基因和草鱼凝血因子FⅦ转录组数据得到草鱼补体因子C5-1基因,cDNA长度3078bp,位于5’端,该序列编码1019个氨基酸,为不完整ORF框,有起始子蛋氨酸M,但无终止子。RACE法扩增得到草鱼凝血因子FⅦ全长cDNA1480bp,开放阅读框1311bp,编码437个氨基酸。草鱼凝血因子Ⅶ基因与补体因子C5-1基因进化方向都与物种进化方向一致,且在结构上高度保守。更多还原

【Abstract】 Hemorrhage of Grass Carp, Started from the1950s, which caused by Grass carp hemorrhage virus(GCHV), become Grass carp breeding development bottleneck, Seriously hinder the development of aquaculture grass carp. Though made some achievements in etiology、Virus biological purification、vaccine and immune gene, but little is known about the gene related to GCHV. Transcriptome analysis can provide a comprehensive understanding of molecular mechanisms involved in specific biological processes and diseases from the information on gene structure and function, which suitable for grass carp resistance of the virus bleeding related research.Use grass carp infected GCHV after Oh、24h、48h、72h、96h、138h. and five tissues of thymus/gill/liver/spleen/head of the kidney to real-time PCR, obtained some Innovative research results.1、Transcriptome database of grass carp spleen after infected with GCHVTake two samples injected after72hours for experiment, one infected GCHV virus and another is infected normal saline. Each of2G data volume and4G in all. Obtain109077unigenes, include108092expression difference unigenes. log2Ratio≥1as the threshold to judge the significance of gene expression difference, and32621unigenes were expression difference of all unigenes, include expression up gene20825and down gene11796.2060unigenes can’t be Predicted protein Coding Region, it can be see as the new gene.2、Selected2signal pathways closely related to GCHV32pathways related to immune and disease-resistant were obtained, include pathways which FDR<0.05and4pathway were related to immune such as complement and coagulation cascades pathway、cytotine-cytotine receptor interaction pathway、 phagosome pathway and hematopoietic cell lineage pathway, the most difference express is complement-coagulation cascades pathway and cytotine-cytotine receptor interaction pathway.3、Choiced9signal genes closely related to GCHV and verified their Periodic express rule after infected GCHV After real-time PCR and incorporation with Grass carp bleeding symptoms choose C3、C5、XCR1、F7、F2、PLG、NGFR、CCR4、XCR1、X nine gene maybe the most related to resist GCHV.4、Cloned firstly C5-1and FⅦ gene cDNA of grass carpComplementary factor C5-1gene cDNA of grass carp is3078bp, encoding1019amino acid, include methionine, but no terminator. The gene full-length cDNA sequence of blood coagulation factor FⅦ from grass carp was cloned by RACE-PCR technique. The results showed that the length of FVE gene cDNA sequence was1480bp, encoding a peptide of437amino acid residues(aa). The result surveyed that evolvement of gene with FⅦ and C5are the same with spicies, and the two gene amino acid structure is highly conservation.更多还原