棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

对棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组中EcoRI-N片段进行序列分析,获得了完整的解螺旋酶基因(hel),其开放阅读框大小为3 762bp,编码一个分子量为146kD的蛋白质.在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在-112位和-189位存在两个TATA box,但未发现早期转录信号CAGT.其在终止密码子下游第12位有一PolyA终止信号AATAAA.在其它真核或原核解螺旋酶中存在的7个保守基元(I、Ia、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ),只有5个(I、Ia、Ⅱ、Ⅲ、Ⅳ)在杆状病毒中保守.同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒(Spodoptera exigue MNPV,SeMNPV)的解螺旋酶具有最高的同源性(66%),与Xestia c-nigrum颗粒体病毒(XcGV)解螺旋酶的同源性最低(43%).HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因.     

利用超氧化物歧化酶增加棉铃虫病毒的感染性

Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HaSNPV) has been used as a commercial pesticide for the control of cotton bollworm. However, some kinds of phenolase (particularly peroxidase) secreted by cotton glands, which generate free radicals, diminish the infectivity of baculoviruses significantly on cotton leaves. Superoxide dismutase (SOD) acts on the polyphenolic substance and eliminates free radicals. Results in this research indicated that SOD-additive HaSNPV has a significantly higher efficacy to kill the bollworm on cotton leaves than HaSNPV alone. Therefore,SOD can be a promising enhancer for the HaSNPV pesticide.     

利用超氧化物歧化酶增加棉铃虫病毒的感染性

棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus,HaSNPV)是棉铃虫的专一性病原,具有宿主特异性高、不影响天敌、不污染环境、对人畜安全等优点,在我国作为商品生物杀虫剂已有10余年的历史.     

Evaluation of Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) for Control of Helicoverpa armigera (Lepidoptera: Noctuidae) on Citrus in South Africa

A concentration of 1.15×10⁷ OBs/mL of HearNPV, sprayed using a knapsack, resulted in a 100% reduction in Helicoverpa armigera larval infestation within 7 days on tomato plants in a hot house environment. A 10-fold lower concentration, 1.15×10⁶ OBs/mL, resulted in a 100% reduction within 16 days. These two concentrations reduced damage to tomatoes by 81 and 69%, respectively. In two field trials on navel oranges, the lower concentration and an additional even lower concentration of 7.26×10⁵ OBs/mL, both resulted in a 100% reduction in H. armigera infestation within 14 days or longer. H. armigera damage to fruit was reduced by up to 84 and 75%, respectively, in the two trials. Rejection for export was reduced by 96 and 62%. This could imply a saving of US$339 per hectare compared to the untreated control. These results were better than those achieved with Bacillus thuringiensis and various standard chemical insecticides used in the same trials. Reasons for these results are discussed.     

中国棉铃虫核多角体病毒基因组XbaI—I片段的序列分析

报道了棉铃虫单核衣壳核多角体病毒 (HaSNPV)XbaI I片段的序列结构。该片段在基因组上定位于 18.4～2 2 .8m .u .,包括 8个完整的开放阅读框架和 p47的 5′端部分序列 :其中ubiquitin ,39K/pp31,lef - 11,p47,Ac34 ,Ac38和Lsel2 5homologue 7个基因是杆状病毒的同源基因 ,另外两个orf5 0 7和orf10 80是HaSNPV的特有基因 ,且具有典型的早期表达基因启动子的特征 ,经软件分析发现这两个基因推导的产物具有典型的跨膜压结构。序列比较分析表明 ,ubiquitin基因与其它杆状病毒的同源基因有相同的保守区 ,39K/pp31具有和其它杆状病毒同源基因不同的启动子结构     

中国棉铃虫核多角体病毒基因组 Xba I-I片段的序列分析

报道了棉铃虫单核衣壳核多角体病毒(HaSNPV)XbaI-I片段的序列结构.该片段在基因组上定位于18.4～22.8 m.u., 包括8个完整的开放阅读框架和p47的5′端部分序列其中ubiquitin, 39K/pp31, lef-11, p47, Ac34, Ac38和Lsel25 homologue 7个基因是杆状病毒的同源基因, 另外两个orf507和orf1 080是HaSNPV的特有基因,且具有典型的早期表达基因启动子的特征,经软件分析发现这两个基因推导的产物具有典型的跨膜压结构.序列比较分析表明, ubiquitin基因与其它杆状病毒的同源基因有相同的保守区, 39K/pp31具有和其它杆状病毒同源基因不同的启动子结构.     

棉铃虫核多角体病毒p10基因的序列及转录分析

为了利用棉铃虫核多角体病毒（HaSNPV）p10基因的启动子构建重组病毒杀虫剂及表达系统,应用末端测序法和引物步移法测定了p10基因的序列,并应用Northern杂交和引物延伸实验对该基因进行了深入的转录特性分析,HaSNPV p10基因被定位于基因组DNA的115.4kb-115.6kb处,转录方向与多角体基因相反,在其上下游分别发现了p26和p74基因。转录分析结果确定了p10基因是个极晚期基因,其转录从杆状病毒晚期转录保守序列GTAAG的第二个A开始,转录产物大小约为430nt。HaSNPV p10基因最早可检测到的转录是在病毒感染后的20h,随后其转录产物量迅速放大,到感染后72h达到非常高的水平。围康时相分析同时还检测到一个大小为1300nt的产物,推测该产物为p26和p10通读的转录产物。对HaSNPV P10蛋白序列的分析表明,该蛋白第6－44位及第51－65位氨基酸序列处含多个典型的卷曲螺旋七聚体重复结构,两片段间相隔6个氨基酸残基,在该蛋白序列的第20－34位与第51－65位存在L－X（2）－L－X（10）－L的亮氨酸重复。Helical net分析表明,HaSNPV P10的疏水氨式酸分布为两个集簇区,两者互为180度转角。     

棉铃虫核型多角体病毒(HaSNPV)G4株Ha109蛋白的原核表达及抗体制备

根据棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single nucleocapsid nucleopoly-hedroyirus,HaSNPV)G4 株基因组全序列,设计引物,利用PCR方法扩增出开放阅读框(open reading frame,ORF)109编码片段并将其克隆至载体pGEM-T Easy,测序正确后将其亚克隆至原核表达载体pGEX-KG,经IPTG诱导,融合蛋白GST-Ha109在E.coli BL21中以包涵体形式得到高效表达.表达目的蛋白大小为38.5 kD,经SDS-PAGE分离纯化,免疫新西兰大白兔制备了多克隆抗体.抗体经1∶6000倍稀释后用于Western b1otting分析,获得特异性显色信号,为深入研究Ha109的功能奠定基础.     

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 2...     

Partial Functional Rescue of Helicoverpa armigera Single Nucleocapsid Nucleopolyhedrovirus Infectivity by Replacement of F Protein with GP64 from Autographa californica Multicapsid Nucleopolyhedrovirus

Two distinct envelope fusion proteins (EFPs) (GP64 and F) have been identified in members of the Baculoviridae family of viruses. F proteins are found in group II nucleopolyhedroviruses (NPVs) of alphabaculoviruses and in beta- and deltabaculoviruses, while GP64 occurs only in group I NPVs of alphabaculoviruses. It was proposed that an ancestral baculovirus acquired the gp64 gene that conferred a selective advantage and allowed it to evolve into group I NPVs. The F protein is a functional analogue of GP64, as evidenced from the rescue of gp64-null Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV) by F proteins from group II NPVs or from betabaculoviruses. However, GP64 failed to rescue an F-null Spodoptera exigua MNPV (SeMNPV) (group II NPV). Here, we report the successful generation of an infectious gp64-rescued group II NPV of Helicoverpa armigera (vHaBacΔF-gp64). Viral growth curve assays and quantitative real-time PCR (Q-PCR), however, showed substantially decreased infectivity of vHaBacΔF-gp64 compared to the HaF rescue control virus vHaBacΔF-HaF. Electron microscopy further showed that most vHaBacΔF-gp64 budded viruses (BV) in the cell culture supernatant lacked envelope components and contained morphologically aberrant nucleocapsids, suggesting the improper BV envelopment or budding of vHaBacΔF-gp64. Bioassays using pseudotyped viruses with a reintroduced polyhedrin gene showed that GP64-pseudotyped Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) significantly delayed the mortality of infected H. armigera larvae.The envelope fusion protein (EFP) of budded viruses (BV) (30) of baculoviruses is critical for virus entry (attachment and fusion) and egress (assembly and budding) (7, 13, 21). Two types of BV EFPs have been identified in the Baculoviridae family of viruses. The F proteins are similar in structure, but they are very diverse in their amino acid sequences (20 to 40% identity). They are widespread within the baculovirus family (group II NPVs of the alphabaculoviruses and in beta- and deltabaculoviruses) (23) and are thought to be carried by ancestral members (26). In contrast, the baculovirus GP64 homologs are all closely related EFPs (>74% sequence identity) and found only in group I NPVs of the alphabaculoviruses (23). It has been suggested that a gp64 gene was acquired relatively recently by an ancestral virus of the group II NPV, thereby giving these viruses a selective advantage and obviating the need of the envelope fusion function of the F protein (23). A nonfusogenic F homolog (F-like protein), however, is maintained in the genome of group I NPVs, functioning as a virulence factor (9, 17, 24, 32).GP64 and F proteins play similar roles during the baculovirus infection processes, such as virus-cell receptor attachment, membrane fusion, and efficient budding. However, there are striking differences between the receptor usage of GP64 and F proteins as well. These two types of proteins are very different in structure, mode of action, and receptor exploitation. The crystal structure reveals that GP64 belongs to class III viral fusion proteins, with its fusion loop located in the internal region of the protein, and proteolytic cleavage is not required for activation of fusion activity (10). F proteins by contrast share common features of class I viral fusion proteins (12). The proteolytic cleavage of the F precursor (F₀) by a furin-like protease generates an N-terminal F₂ fragment and a C-teminal F₁ fragment. This cleavage is essential for exposing the N-terminal fusion peptide of F₁ and for activating F fusogenicity (8, 36). Although the nature of the baculovirus host cell receptors is still enigmatic, it has been reported that Autographa californica multicapsid nucleopolyhedrovirus (MNPV) (AcMNPV)) and Orgyia pseudotsugata MNPV (OpMNPV), both using GP64 as their EFPs, exploit the same insect cell receptor, while Lymantria dispar MNPV (LdMNPV) with an F protein as the EFP utilizes a cell receptor different from that used by AcMNPV (7, 37). Additionally, in the case of SeMNPV, using competition assays, it was confirmed that the baculovirus F protein and GP64 recognized distinct receptors to gain entry into cultured insect cells (34).Pseudotyping viral nucleocapsid with heterologous EFPs to form pseudotype virions is a valuable approach to studying the structure, function, and specificity of heterologous EFPs. It has been a successful strategy to expand or alter viral host range, i.e., in gene delivery (3). For example, vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus and AcMNPV gp64-pseudotyped HIV-1 exhibit high virus titers and wider tropism (5, 14, 38); the gp64-pseudotyped human respiratory syncytial virus (HRSV) lacking its own glycoproteins is of high and stable infectivity (22); furthermore, pseudotyped lentiviruses with modified fusion proteins of GP64 with targeting peptides (i.e., hepatitis B virus PreS1 peptide, involved in viral attachment) or with the decay accelerating factor (DAF) facilitate the targeting to specific cell types or confer stability against serum inactivation, respectively (6, 19). For the Baculoviridae, a series of pseudotyping studies have investigated the functional analogy between GP64 and F proteins. F proteins of group II NPVs (SeMNPV, LdMNPV, and Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus [HearNPV]) can substitute for GP64 in gp64-null AcMNPV viruses (15, 16). Recent studies indicated that many granulovirus (GV) F proteins, but not F protein from Plutella xylostella GV (PxGV), can rescue a gp64-null AcMNPV (16, 39). These results demonstrated that baculovirus F proteins are functional analogues to GP64. Since it was postulated that GP64 was captured by a baculovirus during evolution (24), one would expect the functional incorporation of GP64 into the BV of an F-null group II NPV. However, the reverse substitution of a group II NPV (SeMNPV) F protein by GP64 failed to produce infectious progeny viruses (35).In this paper, we show that AcMNPV gp64 could be inserted into an F-null HearNPV genome and produce infectious progeny virus upon transfection of insect cells. The infectivity of the pseudotyped virus, however, was greatly impaired, and large amounts of morphologically defective BV were produced. Bioassay experiments indicated that the infectivity of GP64-pseudotyped F-null HearNPV for insect larvae was not reduced, but that the time to death was significantly delayed. These results demonstrate that GP64 alone can only partially complement HearNPV F protein function.     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini-F replicon、一个卡那霉素抗性基因和一个lacZα基因8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内,构建了既能在E.coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid-HZ8).另外将HaSNPV的多角体蛋白基因和P10启动子序列取代pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列,构建插入HaSNPV多角体蛋白基因和 P10启动子序列的HapFastBacPhP10供体质粒.利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上,随后将含有eGFP基因的重组HaBacmid DNA转染至HZAm1细胞内.转染5d后,细胞核内能形成典型的多角体,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光.结果证明我们构建的HaBac to Bcac 表达系统能有效的表达外源基因.     

一种新颖的棉铃虫单粒包埋核多角体病毒表达系统

将含有低拷贝数的mini Freplicon、一个卡那霉素抗性基因和一个lacZα基因 8.6kb的DNA片段经同源重组置换到棉铃虫核型多角体病毒基因组中的多角体蛋白基因内 ,构建了既能在E .coli内复制又可在昆虫细胞内复制形成完整的病毒粒子棉铃虫核型多角体病毒Bacmid(HaBacmid HZ8)。另外将HaSNPV的多角体蛋白基因和P10启动子序列取代 pFastBacDual质粒上的AcMNPV的多角体启动子序列和P10启动子序列 ,构建插入HaSNPV多角体蛋白基因和P10启动子序列的HapFastBacPhP10供体质粒。利用HapFastBacPhP10供体质粒将eGFP基因转位至HZ8的Tn7附着位点上 ,随后将含有eGFP基因的重组HaBacmidDNA转染至HZAm1细胞内。转染 5d后 ,细胞核内能形成典型的多角体 ,在萤光显微镜下观察到细胞内显示出强烈的绿色萤光。结果证明我们构建的HaBactoBcac表达系统能有效的表达外源基因     

Natural Control of Helicoverpa armigera in Cotton: Assessment of the Role of Predation

Irreplaceable mortality of Helicoverpa armigera due to natural enemies was studied in cotton in western Kenya. Field populations of H. armigera were followed in three types of subplots: where crawling predators were suppressed, where both crawling and flying predators were suppressed and where predators were unaffected. Ants were the predominant crawling predators, whereas anthocorids were the predominant flying predators. H. armigera mortality from egg to late larval stage was very high (96.4-99.7%) and was greater in the second than in the first generation. Suppression of the different groups of predators did not increase the density of the pest. It is argued that the high level of background mortality obscured the role of predators. The possible role of host-plant condition on background mortality is discussed.     

A Flexible Approach for the Analysis of Rare Variants Allowing for a Mixture of Effects on Binary or Quantitative Traits

Multiple rare variants either within or across genes have been hypothesised to collectively influence complex human traits. The increasing availability of high throughput sequencing technologies offers the opportunity to study the effect of rare variants on these traits. However, appropriate and computationally efficient analytical methods are required to account for collections of rare variants that display a combination of protective, deleterious and null effects on the trait. We have developed a novel method for the analysis of rare genetic variation in a gene, region or pathway that, by simply aggregating summary statistics at each variant, can: (i) test for the presence of a mixture of effects on a trait; (ii) be applied to both binary and quantitative traits in population-based and family-based data; (iii) adjust for covariates to allow for non-genetic risk factors and; (iv) incorporate imputed genetic variation. In addition, for preliminary identification of promising genes, the method can be applied to association summary statistics, available from meta-analysis of published data, for example, without the need for individual level genotype data. Through simulation, we show that our method is immune to the presence of bi-directional effects, with no apparent loss in power across a range of different mixtures, and can achieve greater power than existing approaches as long as summary statistics at each variant are robust. We apply our method to investigate association of type-1 diabetes with imputed rare variants within genes in the major histocompatibility complex using genotype data from the Wellcome Trust Case Control Consortium.     

棉铃虫羽化高峰期有效积温预测法的校正

为减小年际间气温变化对昆虫有效积温预测误差的影响,以新疆石河子垦区121团棉铃虫Helicoverpa armigera(Hübner)羽化高峰期为例,利用single sine模型分别计算12年2种有效积温范围(10～30℃和10～35℃)的累计有效积温值,并获得其多年平均值,依此进行棉铃虫羽化高峰期预测;通过当年与12年(有效积温>0日期至羽化高峰日期)平均气温之差,对预测误差进行校正。结果表明:当年平均气温与12年平均值差值越大,预测误差也越大;各代直线回归校正模型均达到显著水平(P<0.05);2种有效积温范围下,校正后各代平均预测误差天数均有所减少,对越冬代误差校正效果最优,校正后各代历史符合率分别为83.33%、100%、100%和100%、100%、93.33%。该校正方法能够显著提高预测准确度,尤其适用于年际间棉铃虫发育期间平均气温变化较大的代别和地区,同时可为多种害虫预测误差校正提供了依据。     

A method for reducing error in estimating the effective accumulated temperature of the Helicoverpa armigera eclosion peak

为减小年际间气温变化对昆虫有效积温预测误差的影响,以新疆石河子垦区121团棉铃虫Helicoverpaarmigera(Hübner)羽化高峰期为例,利用single sine模型分别计算12年2种有效积温范围(10～30℃和10～35℃)的累计有效积温值,并获得其多年平均值,依此进行棉铃虫羽化高峰期预测;通过当年与12年(有效积温＞0日期至羽化高峰日期)平均气温之差,对预测误差进行校正.结果表明:当年平均气温与12年平均值差值越大,预测误差也越大;各代直线回归校正模型均达到显著水平(P＜0.05);2种有效积温范围下,校正后各代平均预测误差天数均有所减少,对越冬代误差校正效果最优,校正后各代历史符合率分别为83.33％、100％、100％和100％、100％、93.33％.该校正方法能够显著提高预测准确度,尤其适用于年际间棉铃虫发育期间平均气温变化较大的代别和地区,同时可为多种害虫预测误差校正提供了依据.     

Vip3A is responsible for the potency of Bacillus thuringiensis 9816C culture supernatant against Helicoverpa armigera and Spodoptera exigua

Culture supernatant of Bacillus thuringiensis 9816C had high toxicity against Helicoverpa armigera and Spodoptera exigua. However, it lost insecticidal activities after being bathed in boiling water for 5 min. Acrystalliferous mutants of Bt9816C (Bt9816C-NP1 and Bt9816C-NP2) cured of its endogenous plasmids no longer possessed vip3A gene and toxicity. The 89 kD protein which existed in Bt9816C supernatant disappeared in the two mutants' supernatant; nevertheless, the two mutants still exhibited hemolytic and phospholipase C activity as Bt9816C did. The vip3A gene of Bt9816C, vip3Aa18, was cloned and expressed in Escherichia coli BL21. Bioassay demonstrated that the recombinant E. coli had high toxicity against S. exigua. Taken together, it suggested that Vip3A protein was responsible for the toxicity of Bt9816C culture supernatants.