【作者】 赵远；

【导师】 陈克平； 姚勤；

【作者基本信息】 江苏大学 ， 食品科学， 2007， 博士

【摘要】 家蚕核型多角体病毒（Bombyx mori nuclear polyhedrosis virus,BmNPV）是昆虫病毒史上首先发现的。由BmNPV引起的家蚕核型多角体病毒病是养蚕业三大病毒病中危害最为严重的一种。该病在世界养蚕业国家常有暴发,传染极强,难以控制,危害最为严重,常造成巨大的经济损失。目前,四川、浙江、江苏、广东等蚕业主产区,每年由BmNPV引起蚕茧损失,约占蚕病总损失的70%～80%。蚕业界学者多年来一直希望寻找抗病基因、阐明抗性机理、培育抗性品种,至目前为止,在很多领域己取得一定的进展,但仍然有很多问题未能解决。本研究利用抗BmNPV品系NB,敏感性品系306建立回交群体BC₁,利用已经构建的SSR分子标记连锁图,进行抗NPV基因分子标记筛选及连锁定位分析,并尝试分子标记辅助育种技术培育新种质。同时,通过构建近等基因系,采用2-D电泳和质谱相结合的技术,研究BmNPV感染途径相关的中肠组织的蛋白质组时空变化图谱,从蛋白质组水平揭示家蚕对BmNPV的应答模式,通过与对照蛋白质组图谱的比较,希望发现一些与抗性相关的特异性蛋白标志物、参与免疫作用相关的蛋白、参与生命活动的必需蛋白以及其他差异蛋白,为BmNPV病的早期诊断、开发防病新途径和抗病育种提供理论基础。主要研究成果如下:1、在两条染色体上筛选出8个与抗NPV基因连锁的分子标记并进行了定位。利用家蚕减数分裂过程中雌性染色体上的基因不发生交换而雄性染色体上的基因发生交换的特点,用高抗BmNPV家蚕品系NB和高敏感家蚕品系306以及2种类型的回交群体为研究材料,即（306×NB）♀×306♂和306♀×（306×NB）♂,分别记作BC₁F和BC₁M,从已经公布的SSR分子连锁图中共选择103个分子标记特异引物进行抗BmNPV分子标记筛选。用BC₁F群体22个体在第3连锁群上筛选到5个与抗性基因KN1连锁的SSR标记,在第25染色体上筛选到3个与抗性基因KN2连锁的SSR标记。用BC₁M群体192个个体检测,对这些标记和抗NPV基因进行了定位;在第25连锁群上抗NPV基因位于10.8cM处,最近的引物为LFL0442,排列顺序LFL0442、KN₂、LFL1125和LFL0771;在第3连锁群上抗NPV基因位于45.3cM处,最近的引物为LFL0454,排列顺序LFL0542、LFL0773、LFL1148、KN₁、LFL0454和LFL0326。2、采用构建近等基因系的方法创建新种质。利用分子标记辅助育种原理,采用连续回交方法将抗BmNPV基因导入到多丝量优良品种C₀₃中。选择采用常规接种病毒和分子标记辅助育种技术,经过6代回交、接种选择,3代自交,获得对抗BmNPV基因的家蚕新种质“新月”,其经济性状与C₀₃已经接近。实验表明通过分子标记育成的新种质与通过常规育种育成的品种经济性状相差很小,但是通过分子标记辅助选择时间短,规模小,通过检测后代SSR标记,抗性基因留下的可能性更大。3、利用二维电泳技术和质谱技术,在五龄起蚕接种NPV后不同品种不同时间段接种和对照的家蚕中肠组织蛋白表达图谱中筛选出36种已知蛋白并对部分蛋白功能进行分析。以高抗BmNPV的NB品系,高感品系306及以NB、306杂交和回交构建的近等基因系BC8为研究模型,采用蛋白质双向电泳和质谱联用技术,对模型中3个材料,采用接种BmNPV和对照两种组合方式,在五龄起蚕接种NPV后24h、48h、72h进行中肠组织的蛋白差别分析,获得了家蚕三个品种在三个阶段对BmNPV在蛋白质水平上的差异表达图谱。从同一品种同时间段、同一品种不同时间段、不同品种同一时间段对NPV入侵家蚕中肠组织蛋白差异表达图谱进行讨论。选取差异蛋白点胶内酶切后质谱鉴定,对肽指纹图谱在NCBI蛋白数据库里搜索,在18块凝胶上切取了具有明显差异蛋白点256个,通过质谱分析,得到64个点通过数据库比较获得比较满意（得分大于79,或者比对与家蚕同源蛋白,有部分是相同的蛋白）的结果,即是该蛋白的可靠性大于99.5%,利用MASCOT软件对这些结果进行比对分析,共鉴定出了36种已知蛋白质。可能与NPV抗性相关的蛋白:精氨酸激酶、丝氨酸蛋白酶抑制剂、天门冬氨酸氨基转移酶、硫醇抗氧化酶。可能参与免疫作用相关的蛋白:烯酰CoA水合酶、磷酸丙糖异构酶、热休克蛋白。参与生命活动必须的相关蛋白:二氢硫辛酰胺脱氢酶、磷脂酰乙醇胺、型H—ATPase、凝乳蛋白酶抑制剂CI-8A、延胡索乙酰水解酶。其他蛋白:类IDGF蛋白、DNA解旋酶、原肌球蛋白、转录延长因子蛋白、肌动蛋白解聚因子等等。更多还原

【Abstract】 Bombyx mori nuclear polyhedrosis virus(BmNPV),the insect virus first discovered,causes nuclear polyhedrosis disease in Bombyx mori, which is the most serious of three viral diseases in sericulture.The disease often breaks out in sericultural countries,and due to its high infectivity,it is difficult to control,thus resulting in enormous economic loss.Currently, cocoon loss caused by BmNPV accounts for 70%-80%of the total loss caused by silkworm diseases in major sericultural areas,such as Sichuan, Zhejiang,Jiangsu and Guangdong.For years,scientists in sericulture have been striving to search for resistant genes and elucidate the resistance mechanism as well as to breed resistant races.So far,great progress has been made in many fields.However,many problems remain unsolved.Using backcross population BC1,established with strain NB(resistant to BmNPV) and strain 306(susceptible to BmNPV),and the constructed linkage map of SSR molecular marker,this study aimed to screen molecular markers of the genes associated with NPV resistance and make linkage mapping analysis as well as to establish new germplasms employing molecular marker-assisted breeding technique.Furthermore, near isogenic line(NIL) was established,a combination of 2-dimensional electrophoresis and mass spectrum was adopted to study the changes in expression patterns of BmNPV-infection related proteins in midgut tissue at different time points post inoculation and to reveal the response pattern of Bombyx mori to BmNPV at the proteomic level.Through comparison with the expression patterns of the control,a number of infection-related protein markers and resistance-related proteins were discovered,providing theoretical basis for the early diagnosis of BmNPV disease and developing new ways of preventing the disease as well as for breeding resistant races. The major results achieved are as follows:1.Eight molecular markers linked with resistant genes were screened out on the two chromosomes and then located.Based on the fact that,in the meiosis of Bombyx mori,while there is no crossover between genes on female chromosome there will be crossover between those on male chromosome,we used the following strains as research materials:strain NB, which is highly resistant to BmNPV;strain 306,which is highly susceptible to BmNPV;and two types of backcross populations,namely, (306×NB)♀×306♂and 306♀×(306×NB)♂,designated as BC1F and BC1M,respectively.103 molecular marker specific primers were designed according to the published SSR molecular linkage map for screening molecular markers associated with BmNPV resistance.By using 22 individuals of BC1F population,5 SSR markers linked with resistant gene KN1 were screened out on the third linkage group,and 3 SSR markers linked with resistant gene KN2 were screened out on Chromosome 25,and then 192 individuals of BC1M population were used to locate these markers and resistant genes,revealing that the resistant gene on Chromosome 25 was located at 10.8 cM,with the nearest primer being LFL0442 and arrangement order being LFL0442,KN2,LFL1125 and LFL0771,and that the resistant gene on the third linkage group was located at 45.3 cM,with the nearest primer being LFL0454 and the arrangement order being LFL0542,LFL0773,LFL1148,KN1,LFL0454 and LFL0326.2.New germplasm was created by constructing near isogenic line. According to the principle of molecular marker-assisted breeding technique, genes associated with BmNPV resistance were transferred to hypersilkgenous race C03 with good characters through successive backcrossing.Using a routine inoculation method and molecular marker-assisted breeding technique,a new silkworm race called "Xinyue" was obtained after backcrossing for 6 generations,selecting after virus inoculation and self-crossing for 3 generations.This race is resistant to BmNPV and possesses similar economic characters with C03.The result showed that races bred with molecular marker-assisted breeding technique and those bred with conventional breeding techniques have little difference in economic characters.However,molecular marker-assisted breeding technique needs less time and smaller scale,and enables higher possibility of fixing resistant genes due to its ability to detect SSR marker in the progenies.3.Using 2-dimersional electrophoresis and mass spectrometry,36 known proteins were screened out by comparing the expression patterns of proteins in midgut tissue of newly fourth-exuviated larva of different strains at different time points post inoculation with NPV with those of the control newly fourth-exuviated larva,and functional analyses were performed on part of the proteins.With strain NB,which is highly resistant to BmNPV,strain 306,which is highly susceptible to BmNPV,and strain BC8,a near isogenic line,which was established through cross and backcross between NB and 306 as research models,and using a combination of 2-dimensional electrophoresis and mass spectrometry, proteins were extracted from the midgut tissue of silkworms infected with BmNPV first feeding of the fifth instar at 24 h,48 h,72 h post inoculation and from that of the control silkworms,and then analyzed,and differential expression patterns of proteins of the three strains at the three time points post inoculation with BmNPV were obtained.Expression patterns of proteins in the midgut tissue of silkworms infected with BmNPV were discussed among the same strain at the same time point or at different time points,and among different strains at the same time point.After being in-gel digested,the differential protein spots were identified by mass spectrometry,and peptide mass fingerprints(PMF) were used to search NCBI protein database.A total of 256 differential protein spots were cut from 18 gels,and analyzed by mass spectrometry,and 64 of them had satisfactory results(Scores are higher than 79.Or the alignment shows high homology with Bombyx mori proteins with a few of them being identical). That is,the reliability was higher than 99.5%.MASCOT software was used to analyze these results,and 36 known proteins were identified:(1) proteins that may relate to NPV resistance:arginine kinase,thiol peroxiredoxin, serine proteinase inhibitor(Serpin),aspartame aminotransferase(AST); (2)proteins that may relate to immuifaction:Enoyl-CoA Hydratase(ECH), triose phosphate isomerase(TIM),heat shock proteins;(3) the correlated proteins involved in vital movement:lipoamide dehydrogenase, fumary-lacetoacetate hydrolase,phosphatidylethanol-aminesn(PE); H-ATPase;(4) other differential proteins:IDGF- like proteins,DNA helicase,nonmuscl or smooth muscle protein,Translation elongation factor, Actin-depolymerizing factorand so on.更多还原