家蚕浓核病毒中国株基因组DNA的分离纯化与鉴定

家蚕浓核病毒中国株的正链DNA和负链DNA分别包裹在不同的病毒粒子中,而且两条链的大小不同,经高盐浓度的DNA抽提缓冲液提取后,在琼脂糖凝胶电泳谱中呈现出大小不同的两条带,一条6．4kb,另一条5．8kb。作者分别用高盐和低盐抽提缓冲液提取了家蚕浓核病毒中国株的基因组DNA,意外发现用低盐抽提缓冲液抽提出来的基因组DNA在琼脂糖凝胶电泳谱中仅呈现一条带。推测可能是由于大小不同且不完全互补的正负链,在低盐缓冲液中形成有效的互补,因而呈现一条6．2kb的条带。     

家蚕浓核病毒中国(镇江)株结构蛋白的生物信息学比较分析

运用生物信息学方法将家蚕浓核病毒中国(镇江)株的结构蛋白与其它类型家蚕浓核病毒的结构蛋白在理化特性、结构、功能等方面进行了比较分析。结果表明:家蚕浓核病毒结构蛋白是一类稳定的亲水性蛋白,BmDNV-ZJ与BmDNV-2的结构蛋白性质可能比较类似,而BmDNV-ZJ和BmDNV-1结构蛋白序列的理化性参数、序列内部重复片断以及折叠区域差异较大,表明这两种浓核病毒结构蛋白在性状、结构、功能上有较大差异。而BmDNV-ZJ和BmDNV-1结构蛋白序列中有3个不同的LCR功能区域。分子进化聚类分析可得到四大类浓核病毒结构蛋白。     

家蚕二分浓核病毒感染家蚕后的释放动态研究

【目的】研究家蚕二分浓核病毒(Bm BDV)感染家蚕后不同时间段内释放到环境中的病毒量,为通过蚕沙快速检测病毒提供方法和依据。【方法】选取家蚕二分浓核病毒基因组节段VD1上的ns1基因和VD2上的ns3基因中的一小部分片段,分别克隆到p MD18-T载体上,制备标准品质粒,并用实时荧光定量PCR的方法绘制ns1和ns3的标准曲线。通过检测Bm BDV感染5龄家蚕后不同时间蚕沙中的病毒量来衡量释放到环境中的病毒的变化动态。【结果】在病毒感染家蚕第3天,大量的病毒随蚕沙释放到环境中,每微克蚕沙中约含1.38×106个VD1,6.54×105个VD2;从感染后第5天开始,释放到环境中的病毒量呈指数型增加;第7-8天病毒的释放量到达一个平台期,此时每微克蚕沙中约有2.12×107个VD1,1.34×107个VD2。普通PCR结果也反应出了类似的病毒释放动态。【结论】根据Bm BDV感染家蚕后的释放动态,推测病毒的复制周期约60 h。利用实时荧光定量PCR检测蚕沙中的Bm BDV,能简单、快速、准确地诊断病毒的感染。     

家蚕浓核病毒DNV-3（中国株）的VD2基因组序列分析

浓核病毒BmDNV-3(中国株)基因组中含有两种不同的单链线形DNA分子(VD1,VD2)。该病毒的VD2被分离、纯化、克隆到pUC119载体上,并完成了VD2全基因组序列的测定。序列分析显示:VD2全基因组长为6022个核苷酸,末端拥有524个核苷酸反向重复序列(ITRs)。VD2基因组正链含有2个大的开放阅读框,负链含有1个小的开放阅读框。计算机分析推测该基因组正链上开放阅读框(ORF1)及负链上开放阅读框(ORF3)主要编码病毒的非结构蛋白,而正链上开放阅读框(ORF2)主要编码病毒的结构蛋白。比较BmDNV-3 VD2和BmDNV-2(Yamanashiisolate)VD2基因组全序列,两者同源性达97.7%,并且有132个碱基的替代1、1个碱基的删除和2个碱基插入。研究结果显示BmDNV-3 VD2和BmDNV-2 VD2有很近的亲缘关系,但也发生一定的变异,这为更好地理解浓核病毒种类的多样性,也为研究家蚕浓核病毒进化提供了有益的线索。     

创建可视化的家蚕杆状病毒表达系统表达家蚕二分浓核病毒非结构蛋白NS1

重组杆状病毒感染昆虫细胞是表达外源蛋白常用的一种方法。为有效鉴定转染的细胞中是否产生了重组病毒粒子,对质粒pFastBacI进行改造,构建了极早期基因ie1启动子控制的绿色荧光蛋白egfp基因表达盒,以及多角体基因启动子控制的外源DNA的一个通用型双表达载体;通过酶切、连接的方式,将家蚕二分浓核病毒ns1基因连接到多角体启动子下游;在转座酶的介导下,该供体质粒上部分序列可转座到穿梭载体Bm-Bacmid上,进而构建可同时表达egfp和ns1基因的重组杆粒。将构建的该重组杆粒DNA转染BmN细胞,通过观察可视化的绿色荧光信号,可迅速判定转染后的细胞中重组病毒粒子产生的情况,收集转染后的细胞培养上清,将其感染BmN细胞,对感染4 d后的细胞总蛋白进行Western blotting分析,结果表明能杂交到一条36 kDa大小的特异蛋白,表明NS1蛋白成功获得了表达,进而为深入研究ns1基因的功能奠定了基础。     

家蚕浓核病毒 Bm DNV-3(中国株)VD1基因组结构与转录分析

为了进一步认识家蚕浓核病毒BmDNV-3(中国株)VD1基因组的结构和功能,VD1被分离、纯化、克隆到pUC119载体上,完成了基因组全序列的测定。序列分析显示VD1基因组全长为6543个核苷酸,末端拥有224个核苷酸反向重复序列(ITRs)。VD1基因组正链含有3个大的开放阅读框(ORF1-3),负链含有1个大的开放阅读框(ORF4)。比较BmDNV-3的VD1和BmDNV-2(Yamanashiisolate)的VD1基因组全序列,两者同源性为98.4%,并且有107个碱基的替代和1个碱基插入,氨基酸突变集中在VD1ORF3和VD1ORF4。Northern杂交结果显示VD1的左边正链上有1.1kb和1.5kb两个转录本,右边的负链上有一个3.3kb转录本。3′和5′-RACE结果显示1.1kb转录本开始于nt290,结束于nt1437;1.5kb转录本开始于nt1423,结束于nt2931;3.3kb转录本开始于nt6287,结束于nt2922。正链上1.5kb转录本和负链上3.3kb转录本拥有10个核苷酸的3′端的共同序列。研究结果显示该病毒基因转录与已报道的其它浓核病毒存在较大的差异性。     

家蚕浓核病毒(镇江)株主要结构蛋白基因的克隆及表达

家蚕浓核病毒(Bombyx mori densovirus,BmDNV)是一种昆虫细小病毒.与其它昆虫细小病毒感染昆虫体内多种组织不同,家蚕浓核病毒只感染家蚕中肠上皮组织的圆筒型细胞,感染该病毒细胞的细胞核可以被孚尔根和甲基绿浓染,在病毒感染的早期中肠上皮组织细胞数量增加,形成褶皱,最后感染细胞脱落到肠腔中[1-3].自从20世纪70年代末日本学者证实家蚕浓核病是由于家蚕浓核病毒感染引起的以来[4],已经分离得到了多个病毒株系[5-8].根据它们在血清学、理化特性、品种感受性和病理特征等方面的差异,分为BmDNV-1(伊那株)和BmDNV-2(以山梨株为代表)[8-11].     

家蚕浓核病毒 (中国镇江株)在不同感受性宿主体内的复制

家蚕浓核病毒中国镇江株是一株双生浓核病毒(bidensovirus)。其宿主感染后的病症与典型的家蚕浓核病毒(BmDNV-1伊那株)表现相似,病蚕软化,中肠的圆筒型细胞呈浓核症。该病毒的最大特点是基因组中含有二套DNA分子(VD1,VD2),这两种核酸分子以单链( VD1,-VD1, VD2,-VD2)线型方式被分开包装在各自的衣壳蛋白中,成为四种病毒体,而且它自身编码DNA聚合酶。有部分蚕品种对该病毒表现完全抗性,即不发病。分别对敏感性家蚕品种(华八35)和抗性家蚕品种(秋丰d)的幼虫进行经口接种病毒。在接种后,从2h到96h分9个时间点,对中肠组织进行取样。以家蚕细胞质肌动蛋白A3(actinA3)基因作为参比基因,用来标定取样组织细胞数。针对VD1和VD2分别设计特异引物,用荧光定量PCR的方法分别检测各个时间点的样品中的病毒基因组VD1和VD2拷贝数。结果表明:无论是在感性还是在抗性宿主体内,家蚕浓核病毒中国株的基因组VD1和VD2在各时间点拷贝数相近,表现出VD1和VD2是同步复制的;病毒侵入两种宿主中肠的初始量(接种后2h)基本相等,每个细胞约为6~10拷贝数。在敏感性宿主体内病毒感染过程表现为潜伏期,指数增长期,平台期。从接种后2h到12h为病毒潜伏期;12h到36h为指数增长期,倍增时间为1·71h,大约扩增15次;36h到96h为平台期,进入平台期病毒的拷贝数达到20万个。在抗性宿主体内病毒处于一种极低水平的增殖,从添毒后2h的6~10拷贝数到96h的150~200拷贝数,病毒复制倍增时间分别为3h和12h,大约扩增5次。推测家蚕对浓核病毒中国株的抗病性,只是一种慢性的带毒不发病的表现。     

感染家蚕浓核病毒BmDNV1对家蚕抗性和敏感品种生化参数的影响

在家蚕疾病中,病毒性疾病是危害茧产品的主要且最普遍的疾病。病毒性蚕软腐病是由BmIFV, BmDNV1 和 BmDNV2引起的。对印度种质库（Indian Germ plasm stock）中的BmDN V1有抗性和敏感的家蚕品种进行了鉴定。利用标准方法对在BmDNV1感染过程中抗性和敏感品系的主要有机成分的变化（包括总蛋白、碳水化合物和脂类）进行了检测。结果表明： 随着幼虫年龄的增长,对照组和处理组中有机成分（即蛋白质和碳水化合物）的含量也随之增长,但是处理组的增长水平明显地低于各自对照组的增长水平。接种BmDNV1后,与对照组比较,敏感品种体内的血淋巴和中肠组织的总蛋白的量显著地下降。在抗性品种,接种4天后的总蛋白的量有了显著地下降,之后,下降水平小于各自的对照组。在抗性和敏感品种中,血淋巴和中肠组织中的总碳水化合物的量略有下降。在变化不显著的抗性和敏感品种中,血淋巴和中肠组织中的脂质的量有显著地提高。在敏感家蚕品种,生化变化清晰地显示： BmDNV1感染消耗作为主要能量来源的总蛋白和碳水化合物。这些成分的损耗导致了被感染家蚕的生长受阻。     

The genome sequence of silkworm, Bombyx mori.

We performed threefold shotgun sequencing of the silkworm (Bombyx mori) genome to obtain a draft sequence and establish a basic resource for comprehensive genome analysis. By using the newly developed RAMEN assembler, the sequence data derived from whole-genome shotgun (WGS) sequencing were assembled into 49,345 scaffolds that span a total length of 514 Mb including gaps and 387 Mb without gaps. Because the genome size of the silkworm is estimated to be 530 Mb, almost 97% of the genome has been organized in scaffolds, of which 75% has been sequenced. By carrying out a BLAST search for 50 characteristic Bombyx genes and 11,202 non-redundant expressed sequence tags (ESTs) in a Bombyx EST database against the WGS sequence data, we evaluated the validity of the sequence for elucidating the majority of silkworm genes. Analysis of the WGS data revealed that the silkworm genome contains many repetitive sequences with an average length of <500 bp. These repetitive sequences appear to have been derived from truncated transposons, which are interspersed at 2.5- to 3-kb intervals throughout the genome. This pattern suggests that silkworm may have an active mechanism that promotes removal of transposons from the genome. We also found evidence for insertions of mitochondrial DNA fragments at 9 sites. A search for Bombyx orthologs to Drosophila genes controlling sex determination in the WGS data revealed 11 Bombyx genes and suggested that the sex-determining systems differ profoundly between the two species.     

家蚕拟浆细胞具有吞噬功能

目的：通过观察家蚕Bombyx mori吞噬细胞的微细结构,来确定拟绛色细胞是否也具有吞噬功能。方法：用荧光小球微量注射家蚕pnd pS品系的幼虫,经荧光染色剂丫啶橙和碘化丙啶染色循环血细胞后,在荧光显微镜下观察并扫描拍摄。结果：观察发现除颗粒细胞和浆血细胞外,一些原血球细胞（血干细胞）和拟绛色细胞（多酚氧化酶）也能吞噬荧光小球。在拟绛色细胞里还发现许多和颗粒细胞一样的能被丫啶橙染色的颗粒。尽管在小球细胞中没有发现被吞噬的荧光小球,但该类血球有比较多的能被丫啶橙染色的大颗粒,这表明它们可能是已经被吞噬的凋亡小体。结论：除颗粒细胞和浆血细胞外,一些原血球细胞和拟绛色细胞也能吞噬荧光小球。说明拟绛色细胞也具有吞噬功能。     

Purification and characterization of cocoonase from the silkworm Bombyx mori

Cocoonase (CCN) which facilitates the degradation of a cocoon is recognized as a trypsin-like serine protease. In this study, CCN from the silkworm Bombyx mori was purified and comprehensively characterized. Its activity was maximal at about pH 9.8. It was stable above pH 3.4 at 4?°C and below 50?°C at pH 7.5. CuSO₄, FeSO₄, and ZnSO₄ showed inhibitory effects on CCN, but other salts improved activity. Typical trypsin inhibitors inhibited CCN, but the relative inhibitory activities were much lower than those against bovine trypsin. An extract of cocoon shells inhibited trypsin, but it was only slightly inhibitory against CCN. There were significant differences in catalytic efficiencies and substrate specificities as between CCN and bovine trypsin.     

Spindle-E is essential for gametogenesis in the silkworm,Bombyx mori

As a defense mechanism against transposable elements, the PIWI-interacting RNA (piRNA) pathway maintains genomic integrity and ensures proper gametogenesis in gonads. Numerous factors are orchestrated to ensure normal operation of the piRNA pathway. Spindle-E (Spn-E) gene was one of the first genes shown to participate in the piRNA pathway. In this study, we performed functional analysis of Spn-E in the model lepidopteran insect, Bombyx mori. Unlike the germline-specific expression pattern observed in Drosophila and mouse, BmSpn-E was ubiquitously expressed in all tissues tested, and it was highly expressed in gonads. Immunofluorescent staining showed that BmSpn-E was localized in both germ cells and somatic cells in ovary and was expressed in spermatocytes in testis. We used a binary transgenic CRISPR/Cas9 system to construct BmSpn-E mutants. Loss of BmSpn-E expression caused derepression of transposons in gonads. We also found that mutant gonads were much smaller than wild-type gonads and that the number of germ cells was considerably lower in mutant gonads. Quantitative real-time PCR analysis and TUNEL staining revealed that apoptosis was greatly enhanced in mutant gonads. Further, we found that the BmSpn-E mutation impacted gonadal development and gametogenesis at the early larval stage. In summary, our data provided the first evidence that BmSpn-E plays vital roles in gonadal development and gametogenesis in B. mori.     

家蚕3-磷酸甘油脱氢酶基因BmGpd的结构特征与表达谱分析

3-磷酸甘油脱氢酶（GPDH）是真核细胞的烟酰胺腺嘌呤二核苷酸依赖性酶,具有能量传递等重要功能。家蚕Bombyx mori是重要的变态和能量代谢研究模式动物,为了明确GPDH在家蚕中的作用,本研究根据果蝇lethal （2） k05713的蛋白质序列,从家蚕C108品种克隆了2个完整的mRNA转录体,长度分别为3 456 bp和2 979 bp（GenBank登录号为GQ865685和GQ865686）,具有相同的2 166 bp ORF,编码721个氨基酸。克隆拼接了548 bp的第9内含子后,参照大造品种的全基因组数据,推测出BmGpd基因全长27 983 bp（GenBank登录号为EF154335）,包含16个外显子和15个内含子。编码蛋白具跨膜螺旋结构,pI为8.22,分子量为80.5 kD,1~20 氨基酸区域为信号肽序列。分子同源进化和蛋白质基序分析表明,BmGPD是家蚕中一种新GPDH成员,能在大肠杆菌Escherichia coli中诱导大量表达。RT-PCR分析表明,BmGpd基因表达量在5龄中期最高,在3 d的蛹中明显下调,进入滞育时表达量更低;血液中较低,中肠、丝腺、生殖腺和脂肪体中有大量表达。芯片表达谱显示,5龄第3天幼虫头和体壁中表达丰度最高,脂肪体和马氏管中最低,性别差异不显著。EST表达谱显示,4龄第2天中肠中表达丰度最高。RNAi结果显示,家蚕体内存在BmGpd基因产物代谢补偿途径。本研究为深入研究家蚕GPDH的表达调控以及与变态和能量代谢的关系创造了条件。     

Functional analysis of four Gloverin-like genes in the silkworm, Bombyx mori

To identify genes involved in the innate immunity of the silkworm Bombyx mori, we constructed a cDNA library from the fat body of Escherichia coli-challenged B. mori larvae. Based on the expressed sequence tag (EST) data and whole genome shotgun sequence analysis, we found four Gloverin-like genes, BmGlov1-4, in the Bombyx genome. Northern blot and RT-PCR analysis showed that BmGlov1-4 were induced in the larval fat body after an immune challenge by the injection of E. coli; however, less induction was observed after the injection of a yeast Candida albicans. In silico sequence analysis revealed the presence of a motif homologous to NF-kappaB binding site in the upstream region of each BmGlov gene. Moreover, we expressed recombinant BmGlov1-4 proteins using the baculovirus expression system, and found that all the recombinant BmGlov1-4 significantly inhibited the growth of E. coli.     

Characterization of Argonaute family members in the silkworm,Bombyx mori

Abstract The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5′ end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.