【作者】 刘晓勇；

【导师】 陈克平； 姚勤；

【作者基本信息】 江苏大学 ， 食品科学， 2009， 博士

【摘要】 家蚕核型多角体病毒是引起家蚕病毒严重感染的关键病原之一,病毒与宿主的相互博弈一直是研究者们关注的问题。本论文主要从蛋白质组水平研究杆状病毒的ODV病毒粒子结构组分。通过构建近等基因系研究宿主对抗病毒感染的相关蛋白,希望筛选出一些与家蚕抗性或者感性相关的节点蛋白。同时也从DNA水平筛选抗性相关的分子标记。此外,论文中对构建的近等基因系从不同角度进行了评估,对利用家蚕EST数据库鉴定质谱结果也进行了分析。主要结果如下:通过蔗糖密度梯度离心得到了家蚕核型多角体的ODV病毒粒子,并首次使用SDS-PAGE,二维电泳和质谱技术鉴定了ODV病毒粒子或与之相关的蛋白。鉴定了可靠的19种蛋白,包括15种BmNPV编码的病毒粒子主要蛋白,分别是Polyhedrin,P78/83,BmOrf 40,BmOrf 54,GP41/P40,P95,VP39,P25,ODV-EC43,BmOrf 94,BmOrf 109,P74,P49,ODV-E56和PTP,和4种家蚕蛋白,热休克同源蛋白,Hsp21.4,谷胱甘肽硫转移酶,肽基脯氨酰异构酶。其中2种BmNPV的蛋白BmOrf40和PTP,为首次鉴定为ODV组成。并且我们提出一种假说,本研究鉴定的家蚕宿主来源的蛋白可能对ODV核衣壳包装和成熟有着重要的作用。在抗核型多角体病毒（NPV）病的家蚕品种NB和高敏感品种306及其近等基因系BC₉中,采用500个RAPD随机引物分别在各个品系的DNA混合物进行扩增以筛选分子标记,获得了与家蚕抗NPV病有关的分子标记三个OPF-07₂₀₂₃,OPJ-13₁₃₀₀,OPM-16₁₂₀₀。其中OPF-07₂₀₂₃标记通过回交代检测,证明获得的标记真实、可靠,并对该分子标记的片段进行了克隆、测序。通过对该片段的生物信息学分析,推测在此分子标记处可能有DNA片段的插入,还在家蚕Z染色体上发现了一个逆转位子的编码序列。另外还介绍了一种PCR单一产物直接染色判断有或无的定性检测方法。为了利于研究NB品系家蚕这种突出的抗病毒特性相关的蛋白,我们对高抗,高敏感及近等基因系家蚕五龄起蚕中肠、血淋巴和脂肪体组织的蛋白质表达进行了二维电泳分析,并利用质谱对差异蛋白进行鉴定。对3个家蚕品系的差异蛋白表达进行了比较。在中肠中鉴定了5个差异蛋白,其中1和2在抗性品系NB和BC₉表达量高于306,而3,4,5号这3种蛋白在306中表达较高,这些蛋白可能与家蚕中肠对BmNPV的抗性或感性有关。在血淋巴中,有两种差异表达的蛋白,分别是β-N已酰氨基葡糖苷酶2和氨基酰化酶,我们推测在抗性家蚕的血淋巴中过量表达的β-N-乙酰氨基葡萄糖苷酶可能会干扰细胞膜上的GP64蛋白的N连接聚糖,从而阻止了启动二次感染的一个关键蛋白的功能,进而减少了产生有感染力病毒粒子。在脂肪体组织中鉴定了4种蛋白,其中2种可能与能量代谢有关,其中1号蛋白与磷酸甘油激酶（PGK）在序列上高度相似,2号蛋白是家蚕精氨酸激酶（AK）。第4号蛋白可能和整合酶的功能相似,第3号蛋白未能鉴定。为了从分子水平上评估我们构建的近等基因系的可靠性,尤其是对其与感性的轮回亲本之间遗传背景的相似性进行了评估。在本研究中,我们对随机多态性DNA和双向电泳的一些结果进行了分析,证明其相似性虽比理论的要低,但这个模型方法对研究抗性仍然是有价值的,对研究家蚕其它的差异基因也是有借鉴作用的。我们还利用收集的家蚕EST数据经转换格式后形成MASCOT软件识别的数据库,每个EST经6种阅读框翻译,然后通过MASCOT软件对质谱数据进行分析。还举例说明这种方法能鉴定那些在目前的蛋白序列数据库中没有的多肽。这对一些蛋白数据不够充分的物种蛋白鉴定也有参考价值。更多还原

【Abstract】 BmNPV（Bombyx mori L.nucleopolyhedrovirus） is a key pathogeny to silkworm with highly infective.And the reciprocity between virus and host is an important issue which was paid many attentions by researchers all the time.In this paper,we stuied the protein composition of baculovirus ODV on proteomic level,and the related proteins of resistance to infection in host through constructed near-isogenic line,wish to find some proteins which are related to resistance or susceptibility of silkworm.We also screened the molecular makers that related to resistance on DNA level.In addition,we evaluated the constructed near-isogenic line from different points of view,and analyzed the method that identified mass spectrometry results using EST database.The results are shown as follows.We obtained the occlusion-derived virus（ODV） virion of Bombyx mori nucleopolyhedrovirus（BmNPV） by sucrose density gradient centrifugation,and used techniques of SDS-PAGE,2-DE and MS to identify the proteins present within or associated with ODV virion for the first time.19 proteins,including 15 major proteins of the virion encoded by BmNPV,Polyhedrin,P78/83,Orf 40,Orf54,GP41/P40,P95,VP39,P25,ODV-EC43,Orf94,Orf109, P74,P49,ODV-E56 and PTP,respectively,and 4 proteins of silkworm,heat shock cognate protein,heat shock protein hsp21.4,glutathione S-transferase 2 and cyclophilin A,were identified with confidence.Two BmNPV proteins, orf40 and PTP,those didn’t detected as components of ODV previously,were identified in this study.And we propose a hypothesis that those host-derived proteins maybe important for ODV capsid assembly and maturation.In our study,two silkworm strains,namely NB which is highly resistant to NPV and 306 which is highly susceptible to NPV,and their near-isogenic line BCg,were used.500 RAPD random primers were used to screen molecular markers in DNA mixtures of these strains respectively.Three molecular markers namely OPF-07,OPX-06 and OPX-18 were found linked to major gene resistant to NPV.One molecular marker,OPF-07₂₀₂₃,was validated in some backcrossing generations.We cloned,sequenced and analyzed this special fragment appearing only in the resistant strains.And with bioinformatic analysis, we speculate there is an insertion of DNA fragment,and a retrotransposon gene of the Z chromosome in silkworm was found.In addition,a qualitative method was introduced,which can judge the presentation or not of PCR production by staining directly.To facilitate identification of the proteins in NB that are employed in this prominent anti-virus response,we constructed a near-isogenic silkworm line （NIL） by introgression,which is resistant to BmNPV but is almost genetically identical to the parental strain 306 that is BmNPV-susceptible.In this paper, proteome maps and differences of protein patterns of the midgut,hemolymph and fat body tissues of the highly resistant,highly susceptible and near-isogenic silkworm strains were investigated by two-dimensional gel electrophoresis （2-DE） and matrix-assisted laser desorption/ionization -time of flight （MALDI-TOF） mass spectrometry.A comparison between the different protein expressions of these three silkworm strains led us identified five differently expressed proteins in midgut.Among these protein,proteins 1 and 2 were expressed in NB and BC9 higher than in 306,but proteins 3,4 and 5 were expressed in 306 higher than in others,these protein may involved in the resistance or susceptibility of BmNPV.In hemolymph,there are two differentially expressed proteins,beta-N-acetylglucosaminidase 2 and aminoacylase.We speculate that the excessive expression ofβ-N-acetylglucosaminidase in hemolymph of the resistant silkworm can probably disturb the N-linked glycans of GP64 protein on the cell membrane which is an essential process for initiating second infections,and thus reduce the reproduction of infectious viruses.4 proteins were successfully identified in fat body,and 2 of which are possibly related to energy metabolism.Protein 1 was identified similar to phosphoglycerate kinase,Protein 2 was identified with a silkworm arginine kinase.The third one may have similar function as integrase,and the forth one is completely novel.For evaluating the accuracy of Near-isogenic line that we construct on molecular level,especially the high resemblance of heredity background between the constructed near-isogenic strain and the recurrent parent.We summarized the results of RAPD（Random Amplified Polymorphic DNA） and 2-DE（Two-dimensional polyacrylamide gel electrophoresis）,and compared the resemblance between the near-isogenic and parent strains on DNA and protein levels.The results shows,the resemblance level is less than the theoretical value, but still valuable for research of resistance,and for references to other differential genes of silkworm.We collected silkworm EST sequences,each EST sequence was translated in six frames,and the database was transformed to the format which can be recognized by Mascot program.We analyzed the mass spectrometry data through Mascot program,and an example was given to illuminate that this method can analysis novel peptides,which are missing from current protein sequence databases.The result shows this method has some role, and there is reference value to proteins of some species that haven’t enough pepetide data.更多还原