SeMNPV组织蛋白酶基因的克隆表达及其对病毒杀虫的影响

对甜菜夜蛾核型多角体病毒(SeMNPV)的组织蛋白酶(v-cath)基因进行了克隆,序列分析及原核表达.此基因编码区长1014bp,预计编码一个338个氨基酸的蛋白产物,其蛋白的大小约42kD.序列分析表明,SeMNPV的V-CATH与其它杆状病毒的同源蛋白具有相似的保守结构,保留有相同的酶活性位点.根据几种已知的杆状病毒组织蛋白酶序列构建进化树,发现SeMNPV的V-CATH位于NPV组中一个单独的分支,它可能拥有特殊的进化历程.使用1.2μL的表达组织蛋白酶菌液和SeNPV共感染3龄末甜菜夜蛾幼虫,幼虫病毒致死率提高9.21%,病毒致死时间提前12 h,原核表达的组织蛋白酶对病毒杀虫速度有一定的影响.     

SeMNPV组织蛋白酶基因的克隆表达及其对病毒杀虫的影响

对甜菜夜蛾核型多角体病毒(SeMNPV)的组织蛋白酶(v-cath)基因进行了克隆,序列分析及原核表达。此基因编码区长1014bp,预计编码一个338个氨基酸的蛋白产物,其蛋白的大小约42kD。序列分析表明,SeMNPV的V-CATH与其它杆状病毒的同源蛋白具有相似的保守结构,保留有相同的酶活性位点。根据几种已知的杆状病毒组织蛋白酶序列构建进化树,发现SeMNPV的V-CATH位于NPV组中一个单独的分支,它可能拥有特殊的进化历程。使用1.2μL的表达组织蛋白酶菌液和SeNPV共感染3龄末甜菜夜蛾幼虫,幼虫病毒致死率提高9.21%,病毒致死时间提前12 h,原核表达的组织蛋白酶对病毒杀虫速度有一定的影响。     

甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

甜菜夜蛾核多角体病毒(Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析,该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa.将这一ubiquitin基因克隆到原核表达载体pET-28a上,转化至BL21(DE3)中,用IPTG进行诱导表达,对表达的条件进行了优化.用异源的泛素单克隆抗体检测目的蛋白,Western blot实验证明所表达的蛋白是泛素蛋白.同时,我们制备了特异性的抗体,为以后的研究工作做了基础.通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的泛素基因在分子进化上可能有比较独特的途径.     

甜菜夜蛾核多角体病毒泛素基因的克隆及原核表达

甜菜夜蛾核多角体病毒（Spotoptera exigua multi-nucleopolyhedrovirus,SeMNPV)泛素基因ubiquitin被克隆和序列分析,该基因编码区全长243bp,编码80个氨基酸残基,预计蛋白质分子量为9.4kDa。将这一ubiquitin基因克隆到原核表达载体pET-28a上,转化至BL21（DE3）中,用IPTG进行诱导表达,对表达的条件进行优化,用异源的泛素单克隆抗体检测目的蛋白,Western blot 实验证明所表达的蛋白是泛素蛋白。同时,我们制备了特异性的抗体,为以后的研究工作做了基础,通过计算机软件Gendoc对不同来源的泛素进行分析,结果显示,病毒中的泛素与真核细胞中的泛素相比较,泛素的氨基酸序列有较大的变化,杆状病毒的深入素基因在分子进化上可能有比较独特的途径。     

Identification of a hypertrehalosemic factor in Spodoptera exigua

Trehalose is a major blood sugar in insects with a range of physiological functions, including an energy source and a cryoprotectant. Hemolymph trehalose concentrations are tightly regulated according to physiological conditions. An insulin‐like peptide, SeILP1, downregulates hemolymph trehalose concentrations in Spodoptera exigua. Here, we identified a factor that upregulates hemolymph trehalose concentration in S. exigua. Hemolymph trehalose concentrations were significantly increased after immune challenge or under starvation in a time‐dependent manner. To determine endocrine factors responsible for the upregulation, stress‐associated mediators, such as octopamine, serotonin, or eicosanoids were injected, but they did not upregulate hemolymph trehalose. On the other hand, injection with Schistocerca gregaria adipokinetic hormone (AKH) significantly increased hemolymph trehalose concentration in S. exigua. During upregulation of hemolymph trehalose by AKH injection, trehalose degradation appeared to be inhibited because expression of trehalase and SeILP1 were significantly suppressed while that of trehalose phosphate synthase was not significantly changed. Interrogation of a Spodoptera genome database identified an S. exigua AKH‐like gene and its expression was confirmed. During starvation, its expression concentrations were increased, although RNA interference specific to the AKH‐like hypertrehalosemic factor (SeHTF) gene significantly prevented the upregulation of hemolymph trehalose concentrations during starvation. A synthetic peptide of SeHTF was prepared and injected into S. exigua larvae. At nanomolar concentration, the synthetic SeHTF peptide effectively upregulated hemolymph trehalose concentrations. Here we report a novel hypertrehalosemic factor in S. exigua (SeHTF).     

Inheritance of chorionic malformations and insecticide resistance by Spodoptera exigua

Abstract:  Effect of exposure to the organophosphorus insecticide – fenitrothion [O,O-dimethyl-O-(4-nitro-3-methyl)phenyl] – on four generations of Spodoptera exigua Hübner (Lep., Noctuidae) larvae, with regard to the mortality and structure of the chorion of eggs laid was tested. Generations varied in the total mortality rate, although all of them showed some survival rate. The third and the fourth generations showed the lowest mortality among tested ones. Exposure to fenitrothion caused malformations in eggs. The chorion revealed cracks and diminutions of the outer layer. The quality and quantity of malformations increased proportional to the duration of exposure, e.g. the second exposed generation reveald more prominent changes than the first one. Malformations were observed in the next generations, which were not exposed to pesticide. The follicular cells, which are responsible for the structure and sculpture of the eggshell, must have inherited the malforming mechanism. Most probably, malformation takes place during the late development of eggs in the ovarioles.     

稳定表达人SidT2基因细胞系的建立及其dsRNA转运功能的研究

目的:构建稳定表达人SidT2基因的BHK及MDCK细胞系,探讨SidT2基因过表达与细胞转运双链RNA(dsRNA)能力的关系。方法:根据人SidT2基因序列设计引物,克隆其编码区序列,经双酶切后与pEGFP-N3载体连接,构建其真核表达载体,分别瞬时转染BHK及MDCK细胞,并使用G418筛选稳定表达细胞系;在此基础上,体外转录合成绿色荧光蛋白(GFP)dsRNA,以GFP基因为报告基因,进一步分析过表达人SidT2基因对BHK及MDCK细胞转运dsRNA能力的影响。结果:经基因克隆、酶切、连接后,构建了人SidT2基因真核表达载体pEGFP-SidT2;经瞬时转染及G418筛选,获得稳定过表达人SidT2基因的BHK及MDCK细胞系,实时荧光定量RT-PCR分析表明,其SidT2基因转录水平分别提高71、64.5倍;稳定表达SidT2基因后,在培养液中添加GFP dsRNA,GFP荧光强度较对照细胞分别降低88.1%、73.7%,表明稳定表达SidT2基因的BHK、MDCK细胞转运dsRNA的能力显著增强。结论:构建了稳定表达人SidT2基因的BHK及MDCK细胞系,SidT2基因过表达可显著提高外源性dsRNA的转运能力。     

甜菜夜蛾不同龄期幼虫被斑痣悬茧蜂寄生的风险分析

寄生蜂的寄主识别期是构建最优接受寄主模型的重要参数之一。本研究观察了斑痣悬茧蜂(Meteorus pulchricornis)对甜菜夜蛾(Spodoptera exigua)不同龄期幼虫的识别期,并首次用生存分析方法估计寄主被寄生的风险以及寄生经历对寄主识别期的影响。结果表明:在2.5h观察期内,除1龄寄主幼虫外,其他各龄(2~5龄)幼虫均被寄生;对首次寄主识别期的Cox回归模型拟合分析表明,与2龄寄主幼虫(参照虫龄)相比,3~5龄寄主幼虫被寄生的累计风险比率显著高于2龄寄主幼虫(2·6~4·0倍),而对再次寄主识别期的分析未发现3~5龄寄主幼虫与2龄幼虫之间存在显著差异。根据对首次与再次寄主识别期进行的生存曲线比较表明,对2~4龄幼虫的再次寄主识别期显著短于首次寄主识别期;说明寄生经历显著提高了寄生蜂的寄主识别期。由此推测,较高龄期甜菜夜蛾幼虫被无寄生经验斑痣悬茧蜂寄生的风险高于低龄幼虫。     

Identification and recombinant expression of a novel chymotrypsin from Spodoptera exigua

A novel chymotrypsin which is expressed in the midgut of the lepidopteran insect Spodoptera exigua is described. This enzyme, referred to as SeCT34, represents a novel class of chymotrypsins. Its amino-acid sequence shares common features of gut chymotrpysins, but can be clearly distinguished from other serine proteinases that are expressed in the insect gut. Most notable, SeCT34 contains a chymotrypsin activation site and the highly conserved motive DSGGP in the catalytic domain around the active-site serine is changed to DSGSA. Recombinant expression of SeCT34 was achieved in Sf21 insect cells using a special baculovirus vector, which has been engineered for optimized protein production. This is the first example of recombinant expression of an active serine proteinase which functions in the lepidopteran digestive tract. Purified recombinant SeCT34 enzyme was characterized by its ability to hydrolyze various synthetic substrates and its susceptibility to proteinase inhibitors. It appeared to be highly selective for substrates carrying a phenylalanine residue at the cleavage site. SeCT34 showed a pH-dependence and sensitivity to inhibitors, which is characteristic for semi-purified lepidopteran gut proteinases. Expression analysis revealed that SeCT34 was only expressed in the midgut of larvae at the end of their last instar, just before the onset of pupation. This suggests a possible role of this protein in the proteolytic remodelling that occurs in the gut during the larval to pupal molt.     

表达barnase基因的重组棉铃虫核型多角体病毒的构建及其杀虫活性研究

将含有 barnase基因与杆状病毒多角体基因 ( ph)的重组转座载体 p Fb- Bar在大肠杆菌中与含有棉铃虫核型多角体病毒 ( Ha NPV)的穿梭载体 Hanpvid转座并提取重组穿梭载体 DNA转染棉铃虫细胞 ,得到重组棉铃虫病毒 r Ha- Bar.其分子杂交证明 ,昆虫细胞中有 r Ha- Bar的 bar基因转录本存在 ,并能表达产生 33k D的多角体蛋白和 1 2 k D的 barnase.在平板上 ,barnase能降解RNA,出现清晰的降解圈 .r Ha- Bar对三龄棉铃虫幼虫的毒力比野生型 Ha NPV的 LD50 减少 2 0 % ,LT50 减少 30 % .用 barnase的拮抗基因 barstar构建了具有 Neo抗性、并能稳定表达 barstar的棉铃虫转化细胞 AM1 - NB.以携带 barnase基因的重组病毒 r Ha- Bar分别感染转化细胞和正常细胞 ,48h子代病毒在转化细胞中的产量比在正常细胞中高 2 3倍 ,72 h高 1 60倍 .     

Development of a novel expression vector system using Spodoptera exigua nucleopolyhedrovirus

To develop a novel Spodoptera exigua nucleopolyhedrovirus (SeNPV) expression vector system, we examined characteristics of the SeNPV polyhedrin expression in S. exigua cells (Se301). While the extracellular virus titer of SeNPV was 100-fold lower than that of Autographa californica nucleopolyhedrovirus (AcNPV), the levels of polyhedral inclusion body (PIB) formation and polyhedrin expression were higher in SeNPV. To investigate foreign gene expression under the control of the polyhedrin promoter, polyhedrin-based transfer vector pSeKSK2 was constructed, and then recombinant virus SeK1-LacZ was constructed by inserting E. coli lacZ gene as a reporter gene into a genomic DNA of SeNPV using this transfer vector. The beta-Galactosidase activity of SeK1-LacZ in Se301 was about 1.3 times higher than that of BacPAK6. Thus, the SeNPV expression vector system constructed in this study would be very useful in the expression of foreign proteins, specifically for the enhancement of the pesticidal properties of SeNPV by inserting pesticidal genes.     

Nutrient utilization by caterpillars of the generalist beet armyworm, Spodoptera exigua

Abstract.  Beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), caterpillars are destructive crop pests responsible for considerable annual economic losses. These generalist herbivores are challenged with a diversity of dietary choices that can impact their survival, development and fecundity. In the present study, dietary choices of S. exigua caterpillars, based on the protein to digestible carbohydrate (P : C) ratio of the food, and the impact of nutritionally unbalanced foods on caterpillar performance are assessed. In choice experiments, individual third-instar caterpillars are offered simultaneously a P-biased and a C-biased food until pupation. Caterpillars feed nonrandomly and select a slightly P-biased diet (22P : 20C). In no-choice experiments, second instar caterpillars are reared until pupation on diets ranging in P : C ratio from extremely P- to extremely C-biased. High mortality and delayed development are observed on the C-rich, P-poor diets, highlighting the potential deleterious effects of excess carbohydrates and the importance of protein for growth and development. Diet-dependent differences in pupal weight or pupal lipid reserves are not observed. This contrasts with closely-related Spodoptera species where pupal mass and lipid stores increase on C-rich, P-poor diets. On the extremely P-biased diet, performance is similar to that of individuals reared on the self-selected diet, suggesting that these caterpillars may efficiently be deaminating excess amino acids to generate carbon skeletons, which are shunted into lipid biosynthesis. Spodoptera exigua caterpillars exhibit flexible and efficient pre-ingestive nutrient intake regulation and post-ingestive utilization, allowing these generalist feeders to cope with the heterogeneous diets they may encounter.     

Complete genome sequence of a novel picorna-like virus isolated from Spodoptera exigua

The complete genome sequence and the gene organization of a novel insect picorna-like virus, Spodoptera exigua virus (SeV), were determined. The genomic RNA of the SeV was 9501 nt in length excluding the poly(A) tail and contained a single, large open reading frame (nt 392–9424) encoding a 3010 aa polyprotein. Sequence comparisons with other viral polyproteins revealed that the consensus sequences for picornavirus RNA helicase, cysteine protease, and RNA-dependent RNA polymerase (RdRp) proteins are found on the genome in that order from the 5′ to the 3′ end. In terms of sequence similarity, identity, and genome organization, SeV resembled insect picorna-like viruses belonging to the genus Iflavirus. A phylogenetic analysis based on the eight conserved domains in the RdRp sequence showed that SeV was most closely related to the Perina nuda virus and Ectropis obliqua picorna-like virus, suggesting that these three insect picorna-like viruses might share a common ancestor.     

Engineered Repressible Lethality for Controlling the Pink Bollworm, a Lepidopteran Pest of Cotton

The sterile insect technique (SIT) is an environmentally friendly method of pest control in which insects are mass-produced, irradiated and released to mate with wild counterparts. SIT has been used to control major pest insects including the pink bollworm (Pectinophora gossypiella Saunders), a global pest of cotton. Transgenic technology has the potential to overcome disadvantages associated with the SIT, such as the damaging effects of radiation on released insects. A method called RIDL (Release of Insects carrying a Dominant Lethal) is designed to circumvent the need to irradiate insects before release. Premature death of insects’ progeny can be engineered to provide an equivalent to sterilisation. Moreover, this trait can be suppressed by the provision of a dietary antidote. In the pink bollworm, we generated transformed strains using different DNA constructs, which showed moderate-to-100% engineered mortality. In permissive conditions, this effect was largely suppressed. Survival data on cotton in field cages indicated that field conditions increase the lethal effect. One strain, called OX3402C, showed highly penetrant and highly repressible lethality, and was tested on host plants where its larvae caused minimal damage before death. These results highlight a potentially valuable insecticide-free tool against pink bollworm, and indicate its potential for development in other lepidopteran pests.     

甜菜夜蛾的迁徙导致对蛾类生物多样的估计过低

由于蛾类易于采集和鉴定,因而在进行生物多样性调查时,它们成为研究最多的动物类群之一。由于大多数蛾类可以通过灯光诱捕,因此生物多样性研究中普遍采用灯光诱捕法。在抽样样本中,夜蛾科常常所占个体比例最多,该科包括一些著名的迁徙物种,有时捕获的个体数量相当高。本文阐述了2003年发生在托斯卡纳的甜菜夜蛾（Spodoptera exigua）迁徙事件。我们在抽样中发现了大批甜菜夜蛾的标本,从而使这一地区的生物多样性实际值发生了很大改变。因此,我们认为,在对蛾类进行生物多样性调查时,在分析时应该剔除可能涉及到迁徙的物种[动物学报51（5）：909—911,2005]。     

REPAT, a new family of proteins induced by bacterial toxins and baculovirus infection in Spodoptera exigua

Insect larvae spend most of their time eating and the digestive tract is the most crucial barrier for the entrance of many pathogens. In our study, suppression subtractive hybridization (SSH) was used to compare Spodoptera exigua midgut gene expression between larvae exposed to the Bacillus thuringiensis Cry1Ca toxin and non-exposed insects. Based on the SSH results, full cDNA sequences coding for four homologous proteins were obtained. Quantitative and semi-quantitative RT-PCR showed the increased expression of the genes coding for these proteins after exposure to different B. thuringiensis toxins as well as after infection with baculovirus. The proteins were named REPAT after their increased expression in Response to Pathogen. REPAT1, a member of this family, was recombinantly expressed using the baculovirus expression system, revealing the glycosylated nature of the protein. Recombinant baculoviruses expressing REPAT1 were used to infect larvae from S. exigua, showing that expression of REPAT1 was reducing the virulence of baculovirus to the infected larvae. Together, these results suggest a role for REPAT1 in mitigating pathological effects.     

16S rDNA文库法分析甜菜夜蛾中肠共生菌组成

昆虫共生菌在宿主昆虫的生长、发育及适应环境方面具有重要作用。因此,本文利用16S rDNA文库随机测序法分析了甜菜夜蛾Spodoptera exigua(Hübner)初孵幼虫和1日龄整个虫体、3龄幼虫中肠和5龄幼虫中肠的细菌菌群组成,以探究甜菜夜蛾共生菌可能存在的作用。结果显示,在甜菜夜蛾中发现了12种共生细菌,其中有1种共生细菌只能鉴定到伯克氏菌目Burkholderiales;有3种可以鉴定到科,分别是丛毛单胞菌科Comanonadaceae、肠杆菌科Enterobacteria和草酸杆菌科Massilia;剩余8种均可以鉴定到属,分别是不动杆菌属Acinetobacter、噬酸菌属Acidovorax、噬氢菌属Pelomonas、埃希氏菌属Escherichia、柠檬酸杆菌属Citrobacter、肠球菌属Enterococcus、丛毛单胞菌属Cruvibacter和肠杆菌属Enterobacter。在得到的571条有效序列中,不动杆菌属Acinetobacter相对丰度最高,占72.3%,属于优势共生菌。其次是噬氢菌属Pelomonas和噬酸菌属Acidovorax分别占12.8%和5.6%,剩余种类的相对丰度均低于5%。本研究结果证明甜菜夜蛾中存在多种共生细菌,且其种类和相对丰度随甜菜夜蛾生长发育呈动态变化。其中不动杆菌属Acinetobacter作为优势共生菌具有对多种抗生素天然耐药性的重要特征,这为研究甜菜夜蛾为何易产生较强抗药性的机理提供了新思路,也为甜菜夜蛾的微生物防治研究奠定了基础。