【作者】 吕鹏；

【导师】 陈克平；

【作者基本信息】 江苏大学 ， 食品营养与安全， 2014， 博士

【摘要】 家蚕是一种重要的经济和模式昆虫,也是一种重要的可食用资源。随着人们生活水平的提高,家蚕这种优质蛋白质脂肪资源逐渐得到重视,开始出现规模化的利用家蚕或蚕蛹作原料的功能食品生产,这也对蚕业生产提出了新的要求。在蚕业生产中经常遇到一种病毒病的危害,即家蚕核型多角体病毒病(Bombyx mori nucleopolyhedrovirus, BmNPV),每年能对蚕业生产造成巨大危害,约占整个蚕病损失60%以上,在我国夏秋蚕期以及印度等养蚕国家尤为严重,该蚕病目前在世界上还停留在预防阶段。最新的一些研究表明这种杆状病毒能够进入到哺乳动物细胞,这对家蚕作为可食用资源的开发和利用带来一定潜在的威胁。所以无论在食用、生产还是安全方面考虑,对于家蚕核型多角体病毒病抗病基因的寻找和抗性机制的阐明都是一项重要的工作。本课题组从340个家蚕品种资源中鉴定出-种抗性资源,通过组配家蚕遗传模型,从]DNA、RNA和蛋白质水平进行过抗性基因筛选工作,取得一些进展,但到目前为止,没有获得抗性基因。本研究通过第二代高通量测序技术与群组分离分析相结合的方法对BmNPV抗性基因分子标记进行了筛选和连锁分析,最终将抗性基因初步定位,并对抗性基因候选区域进行了分析获得了候选抗性基因,并对一个可能参与抗病毒过程的基因V-ATPase进行了进一步分析。本研究主要结果如下：1.通过第二代高通量测序技术对BmNPV抗性品系NB和感性品系306亲本进行简化基因组测序,一共获得62185个标记,对这62185个标记根据亲本基因型关系进行多态性分析,最终获得5441个多态性标记,其中单核苷酸多态性标记(SNP)4765个,酶切位点单核苷酸多态性标记(EPSNP)355个和插入缺失突变标记(INDEL)311个。2.对于BmNPV抗性家蚕品系NB和感性品系306所具有的5441个多态性标记进行基因结构分析,发现有327个多态性标记存在于基因编码区内,其中有138个为氨基酸水平的突变。通过COG、GO和KEGG数据库对这些突变蛋白进行注释发现,感性亲本306涉及家蚕免疫的蛋白,如丝氨酸蛋白酶(BGIBMGA006406),丝氨酸蛋白酶抑制剂(BGIBMGA013848),胰凝乳蛋白酶(BGIBMGA008242)和一种与人类HSP40/DnaJ同源的热休克蛋白家族成员(BGIBMGA013135),以及两种与DNA复制修复相关蛋白(BGIBMGA001493, BGIBMGA003650)发生了突变,提示306家蚕品系对于各种病原微生物与环境的敏感性很可能与其免疫系统的先天缺失有关。3.对NB和306回交群体BC1按抗感性进行分组,各取40只提取DNA进行混池测序分析,并对筛选到的多态性标记进行连锁分析,最终发现了4个与抗性性状紧密连锁的标记,并将抗性基因定位在了家蚕第23号染色体一段0.67Mb的区域。对于抗性基因紧密连锁区域的分析发现含有22个预测基因,对这22个基因进行RT-PCR检测发现只有6个能在家蚕中肠表达,克隆测序结果表明其中有2个酯酶基因发生突变,功能预测表明很有可能为BmNPV抗性基因。4.对于经口接种BmNPV后抗性和感性家蚕中肠的V-ATPase酶活分析表明,抗性家蚕NB和BC8添毒后24小时中肠内膜系统的V-ATPase酶活出现升高,体外过表达及病毒抑制试验表明V-ATPase确实参与了家蚕抗BmNPV的过程。更多还原

【Abstract】 Silkworm is a kind of important economic and model insect, and it is also a considerable edible resource. With the improvement of living standard, silkworm is generally paid attention due to its high quality of protein and fatty acid, and it is developed to health food to the market successfully. So new quantity and quality demands are set for the sericulture production. In sericulture, the disease caused by Bombyx mori nucleopolyhedrovirus (BmNPV) is the most serious viral infectious disease, which is difficult to control, and bring about60%loss of production in major sericultural areas of the world. In addition, some recent studies showed that the NPV can entry into mammalian cells with high efficiency, which bring some potential safe problems when the silkworm is used as food resource. So the identification of resistance gene and elucidation of the resistance mechanism are important work. Our group identified a local resistant strain NB from344silkworm lines and constructed a BmNPV-resistant near-isogenic line (NIL) BC8. Using this model, some molecules related to anti-BmNPV were discovered in DNA, RNA and protein levels, but the resistant gene is still unknown.In this study, a method of the second-generation sequencing technology combined bulked segregant analysis (BSA) was employed to analyse the molecular markers and linkage analysis of resistance gene, and the rough location of resistance gene was obtained. Some possible resistance genes or related gene were analysed further.The major findings are as fowllows:1. The BmNPV resistant strain NB and susceptive strain306were reduced-representation sequenced by the second-generation high-throughput sequencing.5441molecular markers (SLAF marker) between NB and306were acquired, consisting of4765SNPs,355EPSNPs and311INDEL.2. Through BLAST with the gene and EST libraries of silkDB using5441molecular markers,327markers were found located in the coding sequence (CDS) after corrected manually item by item,138of which lead to mutation in protein level. These mutated proteins were annotated by COG, GO and KEGG database and found that some proteins involved in the innate immune system, such as serine protease (BGIBMGA006406), serpin (BGIBMGAO13848), chymotrypsin (BGIBMGA008242) and a human HSP40/ DnaJ homologous members (BGIBMGA013135) and two proteins related to DNA replication and repair (BGIBMGA001493, BGIBMGA003650) were mutated in306, suggesting that the sensitive to a variety of pathogenic microorganisms and environment of306, is related to its mutated proteins in innate immune system.3. The BSA (bulked segrgant analysis) method was employed to analyse if the maker is linked to BmNPV resistance.6polymorphism markers were confirmed to link to the BmNPV resistance genotype, and4of which were located in the chromosome23. Finally, a0.67Mb region in chromosome23were considered to be the hot region of BmNPV resistance gene, which contain22predicted genes. Through RT-PCR analysis, the expression of6predicted genes could be detected in midgut of silkworm, and the sequencing data showed two carboxylesterase genes were mutated in NB.4. V-ATPase assay of midgut from NB,306and BC8after inoculated with BmNPV showed that V-ATPase activity were up-regulated in the midgut column cells of BmNPV-resistant strains NB and BC8. Besides, the transient over-expression of V-ATPase c subunit in BmNPV-infected silkworm BmN cells could significantly inhibit BmNPV proliferation, indicating that V-ATPase is involved in the silkworm defense response against BmNPV.更多还原