棉铃虫蜕皮调节转录因子在大肠杆菌中的重组表达及其包涵体纯化

蜕皮调节转录因子(hormone receptor 3,HR3)在昆虫蜕皮过程中启动蜕皮相关早期基因簇表达,并抑制蜕皮相关晚期基因簇表达,对昆虫蜕皮级联反应起着关键的调控作用。利用合成的特异性引物通过RT-PCR扩增了棉铃虫 Helicoverpa armigera 蜕皮调节转录因子(HHR3),并与pGEX-4T-1载体连接,在大肠杆菌Escherichia coli DH5α内进行扩增,经过PCR筛选获得了HHR3-pGEX-4T-1重组质粒。 用该质粒转化大肠杆菌表达菌株BL21并进行诱导表达,获得了与谷胱甘肽-S转移酶（GST）融合表达的HHR3包涵体,分子量在94 kD左右,通过无离子去垢剂CAPS（3-［cyclohexylamino］-1-propanesulfonic acid）变性、复性后获得了可溶性GST-HHR3融合蛋白,经凝血酶裂解和SDS-PAGE分离得到纯化的HHR3,经蛋白质N-端测序确认表达正确。用重组表达的HHR3免疫家兔,制备了兔抗HHR3多克隆抗体,免疫印迹检测显示该抗体对HHR3有特异性识别能力, 可以用于HHR3功能与调控等下游研究。免疫印迹检测结果还表明,HHR3在5龄向6龄蜕皮的幼虫脂肪体中高表达,在进入6龄24 h 的幼虫脂肪体中含量明显下降,在6 龄72 h 的幼虫中肠中没有检测到HHR3表达;成虫卵巢中有HHR3表达。     

棉铃虫叉头框蛋白A类似蛋白基因HarmFoxAl的克隆及表达谱分析

【目的】本研究旨在克隆并分析一种棉铃虫Helicoverpa armigera叉头框蛋白A (forkhead box protein A, FoxA)类似蛋白基因HarmFoxAl,探讨2-十三烷酮胁迫下棉铃虫中肠中HarmFoxAl的表达情况,为进一步明确棉铃虫FoxA的功能和参与棉铃虫生长发育的调控通路提供依据。【方法】从棉铃虫幼虫中肠中扩增得到HarmFoxAl的cDNA序列,并对其氨基酸序列和蛋白结构进行分析。将HarmFoxAl的ORF序列连接至pET32a载体并转化大肠杆菌Escherichia coli Transetta菌株,IPTG诱导后检测目的蛋白的表达形式,并利用镍柱亲和层析法纯化融合蛋白。通过qPCR检测棉铃虫不同发育阶段(1-6龄幼虫和预蛹),6龄幼虫不同组织(脂肪体、中肠、体壁和头部)以及10 mg/g 2-十三烷酮处理6龄幼虫不同时间后中肠中HarmFoxAl的表达谱。【结果】HarmFoxAl(GenBank登录号:XM021331806)的开放阅读框为669 bp,编码222个氨基酸,蛋白的相对分子质量和等电点分别为25.03 kD和6.34。氨基酸序列分析表明,HarmFoxAl单体蛋白无信号肽、跨膜区和二硫键,核心区域是由4个α螺旋和3个β折叠组成的球状结构。将重组的Transetta (pET32a-HarmFoxAl)菌株用0.5 mmol/L IPTG在25℃条件下诱导5 h,约45 kD的融合蛋白His-HarmFoxAl能以可溶的形式存在于重组菌中,这与预测的分子量(42.8 kD)相一致。发育阶段特异性表达谱表明,HarmFoxAl在棉铃虫1-3龄幼虫期、6龄幼虫期和预蛹期均有表达,且预蛹期的表达量最高。组织表达谱结果表明,该基因在6龄幼虫的脂肪体、中肠和体壁中表达,且脂肪体内的表达量最高,而在头部中不表达。10 mg/g 2-十三烷酮处理棉铃虫6龄幼虫后中肠中HarmFoxAl的表达量显著降低,但随着时间延长其表达量逐渐升高,处理48 h后表达量显著高于对照。【结论】棉铃虫HarmFoxAl在预蛹期和幼虫脂肪体中表达量最高,2-十三烷酮处理幼虫后HarmFoxAl的表达量急速降低后逐渐升高,推测其在棉铃虫变态发育和解毒代谢过程中发挥重要作用。     

The relative abundance of hemocyte types in a polyphagous moth larva depends on diet

Hemocytes are crucial cells of the insect immune system because of their involvement in multiple immune responses including coagulation, phagocytosis and encapsulation. There are various types of hemocytes, each having a particular role in immunity, such that variation in their relative abundance affects the outcome of the immune response. This study aims to characterize these various types of hemocytes in larvae of the grapevine pest insect Eupoecilia ambiguella, and to assess variation in their concentration as a function of larval diet and immune challenge. Four types of hemocytes were found in the hemolymph of 5th instar larvae: granulocytes, oenocytoids, plasmatocytes and spherulocytes. We found that the total concentration of hemocytes and the concentration of each hemocyte type varied among diets and in response to the immune challenge. Irrespective of the diet, the concentration of granulocytes increased following a bacterial immune challenge, while the concentration of plasmatocytes and spherulocytes differentially varied between larval diets. The concentration of oenocytoids did not vary among diets before the immune challenge but varied between larval diets in response to the challenge. These results suggest that the resistance of insect larvae to different natural enemies critically depends on the effect of larval diet on the larvae’s investment into the different types of hemocytes.     

昆虫蜕皮的分子机理及应用

昆虫蜕皮是一个由PTTH启始的、激素介导的基因序列表达和相互作用的级联反应过程。阐明昆虫蜕皮的分子机理,不仅可以解释发育生物学的科学问题,为害虫控制提供新的思路,还可以从中发现新的可资生产应用的分子。作者通过蛋白质组学方法从棉铃虫Helicoverpa armigera Hubner蜕皮幼虫鉴定到30个差异表达的蛋白质。通过抑制性消减杂交技术,从棉铃虫蜕皮幼虫、变态决定幼虫和5龄取食幼虫鉴定到100个表达序列标签(EST)。证明其中的11个EST在蜕皮或变态时差异表达。通过RT-PCR方法克隆棉铃虫激素接受子3基因,研究该基因在发育中的表达模式。用该基因构建具有绿色荧光蛋白标记和多角体蛋白的基因重组病毒(AcMNPV-GFP-HHR3-Polh)。实验结果表明,AcMNPV-GFPHHR3-Polh病毒可以通过注射或口服感染棉铃虫,导致棉铃虫幼虫非正常蜕皮、生长延缓、半数存活时间下降。该研究显示昆虫蜕皮功能基因在害虫控制中有很好的应用前景。蜕皮功能基因的表达与调控、蜕皮激素介导的信号转导通路、变态过程中组织解体和重建的分子机理、激素调控基因顺序表达的分子机理、变态起始因子、JH受体等是本领域今后的主要研究方向。     

幼虫密度对草地螟血细胞数量和组成的影响

为了阐明幼虫密度对草地螟Loxostege sticticalis L.（鳞翅目： 螟蛾科）细胞免疫能力的影响, 本研究调查了在活体灰菜植株上1,5,10和20头/瓶（900 mL）4种密度条件下的其5龄幼虫血细胞种类、数量和组成。结果表明： 草地螟幼虫血淋巴中有原血细胞、浆血细胞、 颗粒血细胞、珠血细胞和类绛色血细胞等5种（类）血细胞。血细胞总数、 浆血细胞、颗粒血细胞数量随幼虫密度的增加而显著递增, 但原血细胞、珠血细胞和类绛色血细胞数量在幼虫密度间的差异不明显;各种血细胞所占血细胞总数的比例在4个密度中的排序相同, 但10和20头/瓶密度下的浆血细胞比例显著高于1头/瓶的,其余4种血细胞的比例在不同密度之间无显著差异。可见, 幼虫密度主要是通过影响草地螟幼虫浆血细胞和颗粒血细胞的数量及血细胞总数, 从而影响草地螟的细胞免疫能力。     

Effects of high temperature on the hemocyte cell cycle in silkworm larvae

To understand the inhibitory effects of high temperature on insect growth at the cellular level, we investigated the influence of high temperature on the proliferation and division of larval hemocytes in the silkworm, Bombyx mori. Although the total number of hemocytes in the larval body increased enormously over time at 26 degrees C, no increase was observed at 38 degrees C. The number of mitotic hemocytes in circulation increased between days 1 and 2 of the fourth larval stage at 26 degrees C, whereas fewer hemocytes were observed at 38 degrees C. Laser scanning cytometry revealed that the DNA content of hemocytes collected from the fourth-stadium larvae was predominantly 2C, 4C, and 8C, and the proportion of each type of hemocyte changed dynamically with development during the fourth instar. Specifically, the proportion of hemocytes with a higher DNA content increased gradually during the feeding phase then decreased during the molting phase at 26 degrees C; in contrast, no decrease was observed at 38 degrees C. The heat-induced accumulation of 8C hemocytes was mainly detected in granulocytes and plasmatocytes. These data suggest that high temperatures induce a G(2) arrest in larval hemocytes.     

Ultrastructure of the hemocytes of Cetonischema aeruginosa larvae (Coleoptera,Scarabaeidae): involvement of both granulocytes and oenocytoids in in vivo phagocytosis

In the context of comparative studies on immunity defence mechanisms of adults and larvae of the coleopteran Cetonischema aeruginosa (Drury, 1770) the ultrastructure of the circulating hemocytes of the third instar larval stage has been investigated by means of light and transmission electron microscopy (TEM). Six types of hemocytes were found in the hemolymph of C. aeruginosa and they were identified as prohemocytes, granulocytes, plasmatocytes, coagulocytes, oenocytoids and spherule cells. In order to identify the "professional" phagocyte cell, phagocytosis assays were performed in vivo by injection of 0.9 microm carboxylate-modified polystyrene latex beads. It was demonstrated that the granulocytes and the oenocytoids of C. aeruginosa were the only hemocyte types involved in this cellular response.     

Hemocyte differentiation in the hematopoietic organs of the silkworm, <Emphasis Type="Italic">Bombyx mori</Emphasis>: prohemocytes have the function of phagocytosis

Hemocytes isolated from the larval hematopoietic organs of the silkworm were classified following staining with acridine orange and propidium iodide. Among the hemocytes isolated from the hematopoietic organs of whole fifth larval and wandering stages, most were prohemocytes (60%–70%) and oenocytoids (30%–40%). Granulocytes comprised only about 0.5%–1% at the wandering stage and were even rarer at other stages; no spherulocytes or plasmatocytes were found. Therefore, hemocyte differentiation inside larval hematopoietic organs is not as extensive as previously thought. Following 10–30 min in vitro culture of hemocytes isolated from larval hematopoietic organs, many young granulocytes and plasmatocytes appeared. Furthermore, during phagocytosis assays, prohemocytes were seen to adopt the morphology of plasmatocytes, containing fragments of phagocytosed cells. Our results underline the similarities between Drosophila and Bombyx hematopoiesis.     

Characterization of cell clusters in larval hemolymph of the cabbage armyworm Mamestra brassicae and their role in maintenance of hemocyte populations

Maintenance of hemocyte populations is critical for both development and immune responses. In insects, the maintenance of hemocyte populations is regulated by mitotic division of circulating hemocytes and by discharge from hematopoietic organs. We found cell clusters in the hemolymph of Mamestra brassicae larvae that are composed of small, spherical cells. Microscopic observations revealed that the cells in these clusters are similar to immature or precursor cells present in hematopoietic organs. The results of bromodeoxyuridine (BrdU) incorporation experiments demonstrate that these cells are mitotically active. Furthermore, these cells maintain their immature state and proliferate until late in the last larval instar. The results of in vitro experiments showed that most of the cells changed their morphology to one consistent with plasmatocytes or granulocytes, and that the change was promoted by addition of larval hemolymph to the culture medium, in particular when hemolymph was collected at a prepupal stage. Taken together, our results suggested that cells in clusters may be an additional source of hemocytes during larval development.     

Bombyx mori prohemocyte division and differentiation in individual microcultures

We followed the fate of microcultured Bombyx mori prohemocytes in vitro. Prohemocytes isolated from larval hemolymph (day 1 of 4th instar) were maintained for 4-11 days in serum-free MGM-450 medium and some of them underwent mitotic division. Over 60% of the non-dividing prohemocytes differentiated to plasmatocytes or granulocytes. Some of the granulocytes subsequently transformed to spherulocytes. Of the dividing prohemocytes, 59.2% of the daughter cells differentiated into other types of hemocytes such as plasmatocytes, granulocytes and spherulocytes, and the remainder divided into new prohemocytes. Four of these renewed prohemocytes generated daughter cells composed of plasmatocytes and granulocytes. These results suggest that prohemocytes possess the properties of stem cells, and that plasmatocytes and spherulocytes may be terminally differentiated cells, whereas granulocytes, at least in part, may be a transient form of spherulocyte. Oenocytoids were not produced, suggesting that the lineage of oenocytoids differs from that of other types of hemocytes and that it is determined before release from hemopoietic organs.     

苦瓜素Ⅰ对斜纹夜蛾几丁质酶基因(SlCht)和几丁质合成酶基因(SlCHS-A)表达及其生长发育的影响

【目的】几丁质酶和几丁质合成酶对昆虫的变态发育极其重要。本研究旨在阐明苦瓜素Ⅰ对斜纹夜蛾Spodoptera litura几丁质酶和几丁质合成酶基因表达及其生长发育的影响。【方法】利用RT-qPCR检测斜纹夜蛾几丁质酶基因(SlCht)和几丁质合成酶基因(SlCHS-A)在斜纹夜蛾不同发育阶段(卵、幼虫、预蛹、蛹和成虫)和4-6龄幼虫不同组织(体壁、中肠、脂肪体、血细胞、头部和马氏管)中的表达水平以及注射苦瓜素Ⅰ溶液(4 μg/头) 24, 48和72 h时斜纹夜蛾SlCht和SlCHS-A在6龄幼虫各组织中的表达水平。分析在斜纹夜蛾4龄幼虫中注射不同浓度(31.25, 62.5, 125, 250和500 ng/头)的苦瓜素Ⅰ溶液对幼虫和蛹历期、幼虫增重、蛹重、蛹长度、化蛹率、羽化率和存活率的影响,并利用体视显微镜观察斜纹夜蛾幼虫的表型变化。【结果】SlCht和SlCHS-A在斜纹夜蛾中的表达具有发育阶段特异性。SlCht和SlCHS-A在卵期表达量最高,幼虫期和预蛹期的表达量较低;在各幼虫期又表现为6龄幼虫期表达量最高,在其他龄期的表达量低。SlCht和SlCHS-A在斜纹夜蛾6龄幼虫中也显示出组织特异性表达,在血细胞和体壁中高表达,在头部、中肠和脂肪体中低表达。在斜纹夜蛾6龄幼虫中注射苦瓜素Ⅰ能诱导SlCht和SlCHS-A在其各组织中表达量降低;在4龄幼虫中注射苦瓜素Ⅰ后,斜纹夜蛾的生长发育受到抑制,幼虫增重延缓,发育历期延长,化蛹率下降甚至化蛹失败,幼虫及蛹出现较高的畸形率。【结论】苦瓜素Ⅰ可通过诱导SlCht和SlCHS-A表达量的降低来实现对斜纹夜蛾生长发育的抑制作用。本研究为进一步阐明苦瓜素对斜纹夜蛾生长发育的抑制机制提供了新的理论基础,并为进一步应用苦瓜素Ⅰ进行防控奠定了基础。     

Comparative study of hemocytes and associated cells of some medically important dipterans

The aim of this work is to study, characterize, and compare different morphological types of hemocytes of Glossina austeni, G. morsitans, Calliphora erythrocephala, Stomoxys calcitrans, Lucilia sericata, Aedes aegypti, and Culex quinquefasciatus. This information is intended to provide a basis for future studies of the cellular defense mechanisms of these dipterans. Seven morphological types of hemocytes were identified by phase-contrast optics: prohemocytes, plasmatocytes, thrombocytoids, granulocytes, adipohemocytes, oenocytoids, and spindle cells of various sizes. Adipohemocytes are difficult to distinguish from both fat body cells and granulocytes. All seven cell types are not present in every species. For example, thrombocytoids and spindle cells were not found in A. aegypti or C. quinquefasciatus, and oenocytoids were observed only in A. aegypti, C. quinquefasciatus, and in C. erythrocephala. In addition to the hemocytes, fat body cells and nephrocytes are also freely present in the hemolymph of some species but may have gained access to the blood during the bleeding process. With electron microscopy and with thick plastic sections of G. austeni hemolymph, only nephrocytes, plasmatocytes, granulocytes, and spindle cells were identified with any certainty, and the large spindle cells are morphologically very different from those found in the other dipterans. They are rigid cells supported by microtubules running throughout their entire length. These hemocytes are present in large numbers only in newly emerged flies, they are absent in larvae and young pupae, and are rare in old adults. Their disappearance from the hemolymph of newly emerged Glossina appears to be a result of phagocytosis by the plasmatocytes.     

Agglutination and phagocytosis of foreign abiotic particles by hemocytes of the blowely, Calliphora vicina in vivo. I. Dynamics of hemocyte activity during larval development

Three types of Calliphora larval hemocytes have been revealed to be involved in phagocytosis of abiotic foreign particles: thrombocytoids, larval plasmatocytes and plasmatocytes I. Thrombocytoids are the quickest to respond to the appearance of invaders. The onset of test particle entrapment by thrombocytoid cytoplasmic fragments was observed, depending on the larval age within 0.5-5.0 min after injection. Separated fragments were fused, forming strands or roundish agglutinates. Phagocytosis of carbon, carmine or Indian ink particles by larval plasmatocytes occurs far more lately, and no earlier than 20-30 min after injection. Plasmatocytes I are capable of foreign particles adhesion on their surface, with a subsequent morule formation, and of engulfing these particles. These two events start in different time periods: adhesion occurs in 5-10 min, while phagocytosis is observed in 1--3 h. The rate of test particle entrapment and stability of agglutinales clearly depends on the larval age. The most pronounced reaction of hemocytes to foreign particles may be observed by the end of feeding and crop emptying. The second, somewhat less expressed rise of activity occurs in mature larvae not long before the onset of pupariation. Diapause induction is accompanied by reducing activities of both plasmatocytes and thrompocytoids. The importance of different hemocyte types in cellular immune reaction of Calliphora vicina larvae, and co-ordination between plasmatocytes and thrombocytoids are discussed.     

Hemocytes of the blowfly Calliphora vicina and their dynamics during larval development and metamorphosis

On the basis of in vitro observation of live cells and examination of stained slides of larval and prepupal Calliphora vicina hemolymph, seven types of hemocytes have been detected: prohemocytes, stable and unstable hyaline cells, thrombocytoids, spindle cells, larval plasmatocytes, and plasmatocytes I-IV, a. The last representing sequential stages of one cell line differentiation. Prohemocytes are basic cells, from which other forms of hemocytes derive outside the hemopoietic tissue, i.e. in free hemolymph. At the last larval instar, three waves of hemopoiesis occur. Either wave tends to increase the general number of cells and to change the quality of hemocyte population. The first wave occurs at the close of larva feeding and is accompanied by increase in the number of hyaline hemocytes, thrombocytoids and larval plasmatocytes. The second wave of hemopoiesis occurs after the larva's crop emptying. In this period the main increase of hemocyte population occurs at the expense of prohemocytes and plasmatocytes I. The most significant (five-fold) explosion of the population of free hemocytes takes place at the onset of pupariation and correlates with the rise of ecdysone titer. At the first stage of this peak, the amount of plasmatocytes I sharply increases. Further on these are rapidly differentiated into plasmatocytes II and III. After the puparium formation, hemocytes are reduced in number. Plasmatocytes III phagocytose fragments of destroyed larval tissues, pass to the stage of plasmatocytes IV (macrophages), and partially settle on tissues.     

Drosophila Immunity: Analysis of Larval Hemocytes by P-Element-Mediated Enhancer Trap

Our aim was to identify new genes involved in the cellular aspects of defense mechanisms of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.