Gene cloning and expression of cadherin in midgut of Helicoverpa armigera and its Cry1A binding region

Bacillus thuringiensis (Bt), a Gram-positive bacte-rium, produces insecticidal crystal proteins during sporulation. Bt has been used as biopesticides to con-trol a number of insect pests from Lepidoptera, Dip-tera and Hymenoptera and also has become so far the leading gene sources of transgenic plants resistant toinsect pests[1,2]. In China, the use of Bt cotton began in 1997 in Hebei, Shandong and Henan provinces, etc. and rapidly increased to more than 2 million ha in 2002, which is effe…     

Molecular cloning of the phospholipase D gene from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. YU100 and its expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The gene for phospholipase D (PLD) of Streptomyces sp. YU100 was cloned from λ phage library and hetero-logously expressed in Escherichia coli. Using an amplified gene fragment based on the consensus sequences of streptomycetes PLDs, λ phage library of Streptomyces sp. YU100 chromosomal DNA was screened. The sequencing result of BamHI-digested 3.8 kb fragment in a positive phage clone revealed the presence of an open reading frame of a full sequence of PLD gene encoding a 540-amino acid protein including 33-amino acid signal peptide. The deduced amino acid sequence showed a high homology with other Streptomyces PLDs, having the highly conserved ‘HKD’ motifs. The PLD gene excluding signal peptide sequence was amplified and subcloned into a pET-32b(+) expression vector in E. coli BL21(DE3). The recombinant PLD was purified by nickel affinity chromatography and compared the enzyme activity with wild-type PLD. The results imply that the recombinant PLD produced by E. coli had the nearly same enzyme activity as PLD from Streptomyces sp. YU100.     

Additive interaction of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Nucleopolyhedrovirus and Azadirachtin

Mortality of Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) was higher in the combined treatment of Nucleopolyhedrovirus (NPV) and Azadirachtin (AZA) and mortality was increased when AZA concentration was doubled. Larval mortality decreased as the age of the larvae increased in all the treatments. The time for 100% kill of third instar larvae was significantly reduced to 72 h when AZA (0.1 ppm) was combined with NPV (10³ PIB/ml) when compared to 168 and 120 h for the same dose NPV and AZA individual treatments, respectively. The average leaf disc consumption, consumption index (CI), relative growth rate (RGR), the efficiency of conversion of ingested (ECI) and digested (ECD) food values were drastically reduced in the combined treatment of NPV and AZA than in the individual treatments. Larval as well as pupal durations were significantly extended and the adult longevity and fecundity were significantly reduced in the combined treatment of NPV and AZA. Weight of 14 day old control larvae was 420 mg and it was reduced to 299 and 248 mg after NPV (5 × 10² PIB/ml) and AZA (0.025 ppm) treatments, respectively. The larval weight was drastically decreased to 99 mg after the combined treatment at the same dose. The additive interaction between both the treatments, AZA and NPV, was found to be in a dose dependent manner and were highly compatible in disrupting the survival, feeding and biology of H. armigera.     

Predator Presence Moves <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> Larvae to Distraction

Displacement of herbivorous insects by the presence of predators on whole plants has rarely been studied. By semi-continuous observations of an externally feeding insect herbivore and a predator, we show how the mere presence of the predator, Geocoris lubra Kirkaldy (Hemiptera: Geocoridae), on a plant can have a strong influence on the movement and behaviors of Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae. The presence of predators, as opposed to mortality by predators, influenced the proportion of larvae feeding, resting and implementing avoidance activities. In addition, the proportion of time individual larvae allocated to feeding, resting and dropping off plants was affected when predators were present with and without contact between the two. Predators do more than just reduce numbers of herbivores; they influence feeding, displacement and subsequently the distribution of plant damage.     

Putative phosphorylation sites on WCA domain of HA2 is essential for <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> 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 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Tbr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.     

<Emphasis Type="Italic">Rab32</Emphasis> and the remodeling of the imaginal midgut in <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Midgut remodeling is a complex physiological process in holometabolous insects. During midgut remodeling, the larval midgut is decomposed by apoptosis or autophagy during metamorphosis, and the degraded larval midgut is partially absorbed as nutrients by the imaginal midgut for its formation. The molecular mechanism involved in this process is not clear. Here, we found that a Rab protein, which we have named HaRab32, is related to the organogenesis of insect imaginal midgut. Results show that HaRab32 is up-regulated in epidermis and midgut during metamorphosis. Its expression could be up-regulated by 20E. Immunohistochemistry shows Rab32 is distributed in the epithelium of the imaginal midgut during metamorphosis. Knockdown of HaRab32 by RNA interference disturbs the formation of the imaginal midgut. These data imply HaRab32 plays important roles in midgut remodeling by participating in the imaginal midgut formation.     

Inhibiting the Sexual Behavior of Female Cotton Bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

The biological function of a series of aliphatic acids was studied in order to explore a new approach to controlling cotton bollworm Helicoverpa armigera. Calling behavior of the females was observed when they were exposed to one of fourteen aliphatic acids, and mating behavior was studied when females were exposed to one of these five aliphatic acids. Calling behavior and mating rate were reduced significantly when females were exposed to Z6-octadecenoic acid, Z9-octadecenoic acid, 9-octadecenoic acid or Z11-eicosenoic acid. These compounds could be used as female sexual inhibitors, and might be utilized for the control of cotton bollworm in field.     

Interspecific and intraspecific comparisons of ejaculates in the cotton bollworm <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> and the tobacco budworm <Emphasis Type="Italic">H. assulta</Emphasis>

The evolution of reproductive isolation is a prerequisite in the formation of new species. Although there are numerous studies on ejaculates in lepidopteran insects, ejaculate comparisons among sibling species have not been adequately addressed to understand possible reproductive barriers to hybridization. Here, we examined the interspecific and intraspecific variations of ejaculates in the sibling noctuid moths Helicoverpa armigera and Helicoverpa assulta. We found that there were considerable variations in the number of apyrene and eupyrene sperm and the length of eupyrene sperm. Male pupal mass explained not only a significant proportion of the variation in apyrene sperm number in both H. armigera and H. assulta, but also a significant proportion of the variation in eupyrene sperm number in H. assulta. There was a significant positive relationship between the number of eupyrene sperm and the number of apyrene sperm in both species. No difference in the length of eupyrene sperm was found between them; however, ejaculates of H. armigera had many more eupyrene sperm than H. assulta had. In H. armigera, large males generally mated with large females. The evolutionary consequences of these differences are discussed in this paper.     

Synergistic interaction between sublethal doses of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> and <Emphasis Type="Italic">Campoletis chlorideae</Emphasis> in managing <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) is an important solitary larval endoparasitoid of the tomato fruit borer Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) in India. The interaction between Bacillus thuringiensis subspecies kurstaki (Btk) HD-1 and C. chlorideae was studied under laboratory condition to explore their compatibility in managing H. armigera. The results had indicated that the growth and development of H. armigera was affected in a dose-dependent manner upon feeding on sublethal doses of Btk HD-1-treated diets. There were no larval survivors in lethal doses of Btk HD-1 (LC₇₀ and LC₉₀). The growth and survival of the parasitoid were normal when the host larvae were fed with sublethal doses or subjected to short time exposure to lethal doses of Btk HD-1. However, the parasitoid offsprings developed slowly and pupal as well as adult period, adult weight and adult emergence rate were reduced significantly if the parasitoid was developing inside a severely Bt intoxicated host larvae. There were no evident differences in longevity of parasitoid adults that were fed on honey solution containing different concentrations of Btk HD-1 as compared to adults fed only on honey solution. This indicates no direct adverse effect of Btk HD-1 on C. chlorideae. Further, the gravid female parasitoid did not discriminate Btk HD-1 intoxicated and normal H. armigera larvae for oviposition. The result implies that spore crystal formulation of Btk HD-1 can be effectively used in a synergistic manner along with existing natural or prereleased population of C. chlorideae in organic farming or as components in biointensive IPM module for managing H. armigera.     

棉铃虫嗅觉受体基因的克隆及组织特异性表达

昆虫嗅觉受体是一个高度变异的蛋白家族,但是其中一类嗅觉受体很特殊,它们在不同昆虫体内高度保守。该文作者从棉铃虫Helicoverpa armigera体内克隆得到了这类受体基因,命名为ORHarm。ORHarm编码473个氨基酸残基,序列中有7个跨膜区,是典型的G蛋白偶联的受体。ORHarm与已经报道的昆虫同类嗅觉受体的同源性在60％以上,与近缘种烟芽夜蛾Heliothis virescens嗅觉受体的同源性高达99.4％。半定量RT-PCR研究表明,ORHarm主要在棉铃虫成虫触角中表达,在喙中也有表达,但表达量较低,在成虫其他的部位不表达;在棉铃虫发育的各个时期,如卵、幼虫、蛹和成虫体内,也都有表达。ORHarm不仅在感受挥发性气味物质的过程中起着重要的作用,而且也参与液态化学刺激的识别过程。     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Cry1C,Cry2A</Emphasis> and <Emphasis Type="Italic">Cry9C</Emphasis> genes into <Emphasis Type="Italic">Gossypium hirsutum</Emphasis> and plant regeneration

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.     

Cloning and expression of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

Gene cloning and heterologous expression of pyranose 2-oxidase from the brown-rot fungus, <Emphasis Type="Italic">Gloeophyllum</Emphasis><Emphasis Type="Italic">trabeum</Emphasis>

A pyranose 2-oxidase gene from the brown-rot basidiomycete Gloeophyllum trabeum was isolated using homology-based degenerate PCR. The gene structure was determined and compared to that of several pyranose 2-oxidases cloned from white-rot fungi. The G. trabeum pyranose 2-oxidase gene consists of 16 coding exons with canonical promoter CAAT and TATA elements in the 5′UTR. The corresponding G. trabeum cDNA was cloned and contains an ORF of 1,962 base pairs encoding a 653 amino acid polypeptide with a predicted molecular weight of 72 kDa. A Hisx6 tagged recombinant G. trabeum pyranose 2-oxidase was generated and expressed heterologously in Escherichia coli yielding 15 U enzyme activity per ml of induced culture. Structural alignment and phylogenetic analysis were performed and are discussed.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Retroposons of Alu family from <Emphasis Type="Italic">cis</Emphasis>-regulatory modules of <Emphasis Type="Italic">DNAse II</Emphasis> and <Emphasis Type="Italic">CAML</Emphasis> genes affect gene expression in A549 and HEK 293 cells

Promoter fragments of deoxyribonuclease II (DNAse II) and calcium-modulating cyclophilin ligand (CAML) associated with Alu family repeats have been inserted into luciferase reporter vectors. The constructs were introduced into A549 and HEK293 cell lines by transient transfection. Transfected cells were lysed to analyze luciferase activities. It has been shown that Alu repeats inserted into constructs influence the luciferase expression. Therefore, Alu copies associated with cis-regulatory modules in protein-coding genes have biological activity.     

Role of alkaline phosphatase in insecticidal action of Cry1Ac against <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> larvae

Cry1Ac δ-endotoxin produced by Bacillus thuringiensis (Bt) is used as a bio-pesticide for the control of Helicoverpa armigera. Aminopeptidases N (APN) and alkaline phosphatase (ALP) play critical roles in its action against H. armigera larvae. The binding of Cry1Ac with brush border membrane vesicle (BBMV) proteins was increased with the larval development although the sensitivity of larvae to δ-endotoxins decreased. There was higher expression of ALP than APN in early instar larvae with a ~10-fold higher affinity of Cry1Ac towards ALP than to APN. Binding to a specific receptor is therefore more important for the insecticidal activity rather than overall binding to the BBMV proteins. ALP might play a major role in toxicity as compared to APN.