不同世代及中肠病变类型的松毛虫单头幼虫多角体含量

不同世代及中肠病变类型的松毛虫单头幼虫多角体含量许再福（浙江农业大学植保系,杭州３１００２９）戴冠群（华南农业大学植保系,广州５１０６４２）关键词马尾松毛虫,质型多角体病毒,多角体含量,世代,病变中肠源于日本赤松毛虫质型多角体病毒（Ｄｅｎｄｒｏｌｉｍ...     

松毛虫质型多角体病毒的宿主域与交叉感染

自1956年从赤松毛虫Dendrolimus spectabilis上首次发现赤松毛虫质型多角体病毒1型（D. spectabilis cytovirus 1,DsCPV-1）以来,先后从马尾松毛虫D. punctatus、油松毛虫D. tabulaeformis、赤松毛虫、德昌松毛虫D. p. tehchangensis、文山松毛虫D. p. Wenshangensis和落叶松毛虫D. superans上发现了质型多角体病毒(cytoplasmic polyhedrosis virus,CPV)。病毒基因组dsRNA电泳图谱分析表明,这些松毛虫CPV的不同分离株均属于质型多角体病毒1型（cytovirus 1）。这些松毛虫CPV病毒可以感染鳞翅目10科35种昆虫,其中对多种昆虫具有很高的感染力和良好的杀虫效果,可以从中筛选替代宿主生产松毛虫CPV杀虫剂,用于害虫生物防治。松毛虫CPV接种某些昆虫后病毒的基因组dsRNA电泳图谱发生了改变,可能是异源病毒诱发了宿主自身潜伏型病毒的感染复制。     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒。提纯的病毒粒子经SDS－酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10。S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长763bp,起始密码AUG位于3－5残基,终止密码UGA位于747－749残基。推测DpCPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD。和家蚕质型多角体病毒（BmCPV）多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%。     

杆状病毒的垂直传播及绿色荧光蛋白在棉铃虫幼虫中的表达

用转座穿梭系统构建了携带绿色荧光蛋白基因（gfp）的重组棉铃虫核型多角体病毒rHa-FGP,以其多角体添食感染棉铃虫3龄幼虫,室内饲养3代,各代均可见自然光下发绿色荧光的棉铃虫幼虫,其中子代不再重复感染。F₀、F₁、F₂代发绿色荧光的棉铃虫幼虫所占百分比分别为34%、20%、8%。提取虫体内的病毒多角体DNA,以PCR和斑点杂交鉴定表明,gfp不仅在亲代棉铃虫体内正常表达,而且在子代幼虫中表达,HaNPV通过卵实现了垂直传播。     

马尾松毛虫质型多角体病毒多角体蛋白基因的cDNA克隆及序列分析

通过对马尾松毛虫质型多角体病毒的增殖、纯化,获得一株单一类型的质型多角体病毒.提纯的病毒粒子经SDS-酚抽提,琼脂糖凝胶电泳分离基因组dsRNDA,回收纯化第十片段S10.S10经DMSO变性,逆转录合成cDNA第一链,PCR扩增后,克隆在pGEM-T载体上.对重组子进行限制性内切酶分析及序列测定,结果表明,克隆片段全长763bp,起始密码AUG位于3～5残基,终止密码UGA位于747～749残基.推测DpGPV多角体蛋白基因编码248个氨基酸的多肽,分子量28kD.和家蚕质型多角体病毒(BmCPV)多角体蛋白基因相比较,核苷酸和编码氨基酸序列同源性分别为89.3%和97.6%.     

从广西蚕区分离的家蚕质型多角体病毒生物学特征研究

【背景】家蚕中肠型脓病是一种传染性强、危害大的病毒病,其病原为家蚕质型多角体病毒（Bombyx mori cytoplasmic polyhedrosis virus,BmCPV）,该病原抗逆性强、宿主域广,防控难度大。【目的】调查广西蚕区家蚕中肠型脓病发生情况,并研究家蚕质型多角体病毒的感染性、形态特征和分子鉴定,为养蚕生产中有效防控该病提供参考依据。【方法】通过外观和解剖观察病症与显微镜检验相结合,调查养蚕生产家蚕中肠型脓病的发生率;采用生物试验方法测定多角体病毒对家蚕的半数感染浓度（IC₅₀）;利用光学显微镜和扫描电镜观察多角体病毒的外部形态,利用透射电子显微镜观察多角体病毒的内部结构;采用PCR扩增和测序进行分子鉴定。【结果】广西蚕区家蚕中肠型脓病普遍存在,金城江蚕区和东兰蚕区的家蚕中肠型脓病平均发生率分别为6.06%和13.02%,最高发生率达到30.41%;分离获得2株BmCPV病原（暂命名为BmCPV-J和BmCPV-D）,它们的半数感染浓度（IC₅₀）分别为4.88×10³ PIBs/mL和1.63×10⁴ PIBs/mL,都具有很强的致病性;2株BmCPV的形态均为六角形多角体,大小有差异,多角体直径为1.0-3.4 μm;从2株BmCPV内部结构观察到球形病毒粒子,直径为30-50 nm,有刺状突起;可以扩增出BmCPV-J和BmCPV-D的RNA复制酶基因目的片段,BmCPV-J的目的片段序列与BmCPV参考株一致,而BmCPV-D的目的片段序列与参考株有2个碱基的差异。【结论】广西蚕区家蚕中肠型脓病危害严重,该病病原感染力强、分布广,多角体病毒具有典型的质型多角体病毒特征,属于家蚕质型多角体病毒。研究结果为养蚕生产中有效防控家蚕中肠型脓病提供重要依据。     

文山松毛虫质型多角体病毒杀虫剂的研制

报道了文山松毛虫质型多角体病毒杀虫剂的研制,包括多角体的纯化、辅助剂的筛选、剂型研制、产品包装以及病毒杀虫剂的产品质量检测及生产等.该杀虫剂为乳剂,多角体浓度达2.5×109PIB/mL;所选辅助剂取材方便、容易配制,对环境没有污染.经安全检测证明,无致病菌,符合国家卫生标准,对试验动物小白鼠无毒性和致病性.生物测定用1×106PIB/mL感染2龄文山松毛虫幼虫,其死亡率平均为85.5%.     

家蚕质型多角体病毒荧光定量PCR检测方法的建立

根据GenBank报道的家蚕质型多角体蛋白基因的保守序列设计特异性引物扩增118bp核苷酸片段,使用含有目的基因片段的重组质粒标准品绘制标准曲线,建立家蚕质型多角体病毒的实时荧光定量PCR检测方法。结果表明,标准曲线中模板拷贝数(X)与Ct值(Y)关系为Y=-3.582lgX+38.748,相关系数R2=0.999,构建的实时荧光定量PCR检测方法具有良好的敏感性、特异性和重复性,可用于家蚕质型多角体病毒病的快速检验及该病的流行性调查研究。     

文山松毛虫质型多角体病毒杀虫剂的研制

报道了文山松毛虫质型多角体病毒杀虫剂的研制 ,包括多角体的纯化、辅助剂的筛选、剂型研制、产品包装以及病毒杀虫剂的产品质量检测及生产等。该杀虫剂为乳剂 ,多角体浓度达 2 .5×10 9PIB/mL ;所选辅助剂取材方便、容易配制 ,对环境没有污染。经安全检测证明 ,无致病菌 ,符合国家卫生标准 ,对试验动物小白鼠无毒性和致病性。生物测定用 1× 10 6PIB/mL感染 2龄文山松毛虫幼虫 ,其死亡率平均为 85 .5 %     

马尾松毛虫CPV基因组S7的序列分析及部分序列的原核表达

马尾松毛虫质多角体病毒(湖南株)基因组S7节段(AY180908)cDNA克隆及序列分析结果表明S7由1501个碱基组成,编码由448个氨基酸组成的分子量为49.8 kDa的多肽P50.5′末端和3′末端具有5′-AGTAA-3′和5′-GTTAGCC-3′末端保守序列.该基因组与舞毒蛾质多角体病毒1型和家蚕质多角体病毒1型S7节段有很高的同源性,核苷酸序列同源性分别为97.2%和87.0%,氨基酸序列同源性分别为98.7%和92.8%.P50多肽与人型支原体的 DnaK样蛋白在C-末端有相似性.本文报道了编码P50 C259的cDNA序列的克隆并作了原核表达,当用1.0 mmol/L IPTG 诱导2h,分子量约为37.3 kDa的融合蛋白在大肠杆菌BL21中得到大量表达.     

利用黑色斑蚕作亲本、选育日系普斑限性品系

根据家蚕Bombyx moli斑纹互作原理,利用2032限性品种的雌,与自然突变体雄杂交。F2代出现分离,于是淘汰所有黑色斑蚕,只留下普斑蚕和素斑蚕,即得到新限性普斑系。新限性品系得到后,做连续3代的系统选育,其中F3代为蛾区混合育,F4～F5代采用单蛾育。蛾区混合育着重个体选择,单蛾育以蛾区选择为主,个体选择为辅。性状基本稳定后,即初步对其作主要经济性状的测定。结果显示:新限性品系在5龄经过、全龄经过上比两亲本略短。在全茧量、茧层量、茧层率几项指标上较两亲本为优,分别比两亲本平均值提高31%、38%、5%。     

马尾松毛虫CPV基因组第5片段的cDNA克隆及其序列分析

通过差速离心从感染的马尾松毛虫幼虫虫体中提取质型多角体病毒。碱解法提纯病毒粒子,1％琼脂糖凝胶分离基因组dsRNA,回收纯化第五片段(S5)。根据同源性设计五对引物,经RT-PCR,最终获得五个亚克隆片断,测序拼接后,得到S5全长。片断全长2851个核苷酸,包括一个2643个核苷酸的开放阅读框。推测DpCPVS5基因编码了881个氨基酸长的多肽,分子量为100．3kDa,与舞毒蛾质型多角体病毒(LACPV—1)和家蚕质型多角体病毒(BmCPV)比较,核苷酸和氨基酸的同源性都很高。进一步分析,利用几种CPV序列绘制了系统进化树,对病毒的分类和进化做了探讨研究。     

饲料中转基因成分对家蚕生长发育的影响

采用家蚕（Bombyx mori）H9品种作为实验动物,分别用转基因和非转基因大豆粉制作的人工饲料进行饲育。通过饲育试验,比较分析了不同处理后家蚕的龄期经过时间、全茧量、茧层量、茧层率、蛹体重、疏毛率、死亡率、每龄眠起体重等经济性状指标。同时采用PCR方法对转基因饲料饲育后家蚕组织中外源基因的表达情况进行了检测。结果显示,含转基因大豆粉的人工饲料对家蚕的生长发育并无显著影响,且不存在外源基因的污染。     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

QTL mapping of economically important traits in Silkworm (Bombyx mori)

The construction of linkage map is both a funda-mental research area and an important aspect of gene analyses in genetics. It provides the guidelines for breeding. A sound linkage map is also necessary for further genetic analysis. In recent years, great and rapid progress has been made in molecular biology, which enables fingerprinting of organisms at the ge-nomic level. Many molecular marker techniques have been well established. Heartening progress has been made in many organisms in the co…     

Impact of a novel species of Nosema on the southwestern corn borer (Lepidoptera: Crambidae)

A study was undertaken to elucidate the impact of an undescribed Nosema sp. on the southwestern corn borer (SWCB; Diatraea grandiosella Dyar). The Nosema sp. (isolate 506) included in the study was isolated from an overwintering SWCB larva in Mississippi. It was highly infectious per os, with a median infective dose of 2.0 x 10(3) spores per larva. Even at the highest dosage tested (10(7) spores per larva), minimal mortality (< or = 3%) was observed in infected larvae, pupae, and adults reared in the laboratory on an artificial diet. However, infected pupae (0- and 7-d-old) were smaller, and the time to adult eclosion from pupation was slightly increased. Furthermore, the number of eggs produced by infected SWCB female moths substantially decreased (32%), and this effect was most pronounced on day 2, when the greatest number of eggs were oviposited by infected and noninfected moths. For eggs produced by infected females mated with infected males, hatch was slightly decreased by 16 and 15% for eggs laid on days 2 and 3, respectively. In addition, egg hatch was reduced in eggs oviposited by noninfected females mated with infected males on day 3. A low prevalence of infection (< 6%) was observed in the F1 generation originating from infected females mating with noninfected males, from noninfected females mating with infected males, and from infected females mating with infected males. Nosema 506 spores were observed in the proximity of reproductive tissues of infected female and male moths. Spores also were detected on the chorion surface and within eggs laid by infected females. Furthermore, 1-11% of larvae hatching from surface-sterilized eggs were infected by Nosema 506 indicating a transovarial mechanism of transmission.     

QTL mapping of economically important traits in Silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>)

A backcrossed population(BC1)was derived from a cross between C1AFLP technique was employed for mapping the QTLs.The QTLs for the whole cocoon weight,cocoon shell weight,ratio of cocoon shell,weight of pupae etc.Were analyzed and 11 QTLs were detected based on the constructed linkage map.Two QTLs for whole cocoon weight were localized on linkage group 6 and 19; three QTLs for cocoon shell weight were localized on linkage group 3,14 and 19; three QTLs for ratio of cocoon shell were localized on the linkage group 2,11and 15,and three QTLs for the weight of pupae were localized on linkage 2,14 and 19.All these have laid an important base for the marker assisted breeding of the silkworm.     

Oviposition Preferences of the African Wild Silkmoth, <Emphasis Type="BoldItalic">Gonometa postica</Emphasis> Walker (Lepidoptera: Lasiocampidae) on Different Substrates

Gonometa postica Walker (Lepidoptera: Lasiocampidae) is currently being utilised for commercial wild silk production in Eastern Kenya. The oviposition preferences of female G. postica on four substrates in a net-sleeved cage was studied in the laboratory in the long and short rainy seasons in 2007 for two generations, i.e. early (late March to April) and late flights (late September to October) of the moth. The first generation moths laid the highest number of eggs on net sleeves followed by plastic pot, twigs and wooden plank, whereas the second generation moths preferred wooden plank and net sleeves over the plastic pot and twigs. The mean number of eggs, egg clusters laid and percentage egg viability were significantly higher, and the oviposition period was longer for the first than the second generation moths. The mean weight of cocoons was significantly heavier for the first than the second generation. A highly significant positive linear relationship existed between the total number of eggs laid and the cocoon weight. The slopes of the two regression lines for the number of eggs laid against the weight of cocoons were significantly different between the two generations. This information serves to optimise the production of eggs in net sleeved cages in an indoor environment for the semi-captive rearing of the larvae.     

Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility

Digital gene expression (DGE) was performed to investigate the gene expression profiles of 4008 and p50 silkworm strains at 48 h after oral infection with BmCPV. 3,668,437 clean tags were identified in the BmCPV-infected p50 silkworms and 3,540,790 clean tags in the control p50. By contrast, 4,498,263 clean tags were identified in the BmCPV-infected 4008 silkworms and 4,164,250 clean tags in the control 4008. A total of 691 differentially expressed genes were detected in the infected 4008 DGE library and 185 were detected in the infected p50 DGE library, respectively. The expression profiles identified some important differentially expressed genes involved in signal transduction, enzyme activity and apoptotic changes, some of which were verified using quantitative real-time PCR (qRT-PCR). These results provide important clues on the molecular mechanism of BmCPV invasion and resistance mechanism of silkworms against BmCPV infection.