蚯蚓纤溶酶新基因PV242的克隆与表达

从我国特有的双胸蚓属（Lumbricus bimastus）蚯蚓体内提取到一种有纤维蛋白平板溶解活性的蛋白质,采用聚偏二氟乙烯膜(PVDF)微量测序法测得蛋白质的N端氨基酸序列,依此设计简并引物,通过RT-PCR获得其对应cDNA.该cDNA全长888 bp,1～726位核苷酸对应读码框架(ORF),编码含242个氨基酸的成熟肽.终止子（TAG）位于cDNA的727～729位,其余核苷酸序列为3′端非编码区.成熟肽命名为蚯蚓纤溶酶PV₂₄₂.蛋白质预测得知蛋白质等电点为4.33,含组氨酸(His44)及丝氨酸 (Ser191)两个活性位点;蛋白质由两个结构域组成,表面有活性裂隙;该蛋白质属丝氨酸蛋白酶超家族胰蛋白酶类.经国际基因库等多种查询比较未见PV₂₄₂基因的报道,为首次发现的新基因,在国际基因库的输入号为GenBank AF109648.随后构建了pTrxFUS-PV₂₄₂重组质粒,并在大肠杆菌GI724中获得融合蛋白TrxA/PV₂₄₂的可溶性表达,采用离子交换层析法纯化表达蛋白,融合蛋白有纤维平板溶解活性.     

二化螟piggyBac类转座子的克隆与分析

piggyBac转座子是DNA型转座子, 广泛分布于生物体内。基于piggyBac转座子超家族成员IFP2开发的转基因工具载体是目前转基因研究中使用最广泛的载体之一, 因此piggyBac转座子的研究受到广泛的关注和重视。本文是对二化螟Chilo suppressalis内源性piggyBac类转座子（piggyBac-like element, CsuPLE）的首次报道。克隆的CsuPLE（GenBank登录号： JX392388）全长2 537 bp, 包含一个长1 914 bp的完整开放阅读框（open reading frame, ORF）, 编码含637个氨基酸残基的转座酶, 转座酶中含有piggyBac家族保守的“DDD-domain”。CsuPLE全长序列具有完全对称的13 bp反向末端重复序列（inverted terminal repeats, ITRs）以及非完全对称的21 bp内部重复序列（internal repeats, IRs）, 在二化螟基因组上插入在特征性的“TTAA”靶位点重复（target site duplication, TSD）处。在我国地理跨度很大的不同二化螟种群中均存在结构完整的CsuPLE序列。本研究结果为深入研究piggyBac转座子的结构与功能的关系提供了新的素材, 也为评价利用转座子载体系统在二化螟体内进行转基因操作的可行性和安全性提供了重要的理论基础。     

染色质构象调控真核基因的表达

真核基因的表达受到各种顺式调控元件、反式作用因子、染色质DNA以及组蛋白表观遗传修饰等多因素、多层次的调控。染色质三维空间结构的变化在调控真核基因表达方面也发挥了至关重要的作用。染色质构象的变化一方面可以使增强子等调控元件与靶基因相互靠近,从而促进基因表达;同时也可能通过形成空间位阻结构阻碍调控元件作用于靶基因,抑制基因表达。虽然染色质结构变化调控真核基因表达的机制仍缺乏较为精确的分子模型,但在组蛋白修饰、核小体定位、染色体领域以及染色质间相互作用等表观遗传学研究中,已经发现有诸多证据支持染色质构象在真核基因表达调控中的重要地位。文章主要综述了染色质结构及其构象的变化等对真核基因表达调控的影响。     

褐飞虱抗吡虫啉品系生物适合度研究

毒力测定结果显示 ,虽然褐飞虱NilaparvatalugensSt l田间品系对吡虫啉还没有表现出明显的抗性 ,但室内筛选品系已经出现了一定水平的抗性 ,抗性品系 1 (R1)和抗性品系 2 (R2 )的抗性倍数分别为 2 5 3 8和 68 92。通过建立抗性品系、田间种群和敏感品系的生命表 ,发现抗性品系适合度显著下降 ,而且R2 品系下降的幅度明显大于R1品系。存在显著变化的因素有低龄若虫存活率、成虫羽化率、交配率、产卵量和孵化率下降 ,雌虫寿命缩短 ;R2 品系还表现为卵历期延长 ,3龄若虫至 5龄若虫存活率下降。R2 品系产卵高峰期迟 ,高峰期日虫量显著低于敏感品系和田间种群。用种群数量趋势指数 (I)来确定抗性品系的相对生物适合度 ,发现与敏感性品系相比 ,R1品系和R2 品系的相对适合度分别为0 60 9和 0 2 45。     

害虫的基因沉默控制

作为现代分子生物学的重大突破性成果之一,基因沉默是人为调控基因表达的重要途径之一。本文介绍了基因沉默的发现历史、作用机制及其在昆虫中的研究进展,以及利用基因沉默原理进行害虫控制的探索,并展望害虫基因沉默控制的应用前景。     

Clonorchis sinensis infection modulates key cytokines for essential immune response impacted by sex

Infection with helminths can modulate the host immune response, which ultimately shape morbidity and mortality of the associated diseases. We studied key cytokines for essential immune response in sera from 229 southeastern China individuals infected with Clonorchis sinensis and 60 individuals without C. sinensis infection, and measured serum specific IgG and IgE against worms in these people. Individuals infected with C. sinensis had significantly higher antigen-specific IgG and IgE levels, which were positively correlated with egg counts in feces. However, less enhancement of IgE antibody was observed in females when compared to males with similar infection levels. C. sinensis infection caused diminished Th1 cytokines (IL-1β, IL-2, IL-12p70, IFN-γ and TNF-α), Th2 cytokine (IL-4), as well as Th17 cytokine (IL-17A) in sera, which showed decreasing trend by infection intensity. Notably, these phenotypes were more significant in females than those in males. Although C. sinensis infection is associated with the development of hepatobiliary diseases, there was no significant correlation between the dampened cytokine profiles and the hepatobiliary morbidities. Our study indicates C. sinensis infection is strongly related to the immune suppression in human. Sex differences shape the immune milieus of clonorchiasis. This study provides a better understanding of how worms affect immune responses and cause a long-term immune alternation in humans with C. sinensis infection.     

Hydrogen sulfide stimulates β-amylase activity during early stages of wheat grain germination

We recently reported that H₂S could significantly promote the germination of wheat grains subjected to aluminum (Al³⁺) stress.¹ In these experiments seeds were pretreated with the H₂S donor NaHS for 12 h prior to Al³⁺ stress. During this pre-incubation period we observed that H₂S increased the activity of grain amylase in the absence of Al³⁺. Using embryoless half grains of wheat we now show that H₂S preferentially affects the activity of endosperm β-amylase and that α-amylase synthesis and activity is unaffected by this treatment.Key words: α-amylase, β-amylase, hydrogen sulfide, reactive sulfur species, seed germination, wheat (triticum)Cereal grains contain many acid hydrolases, some synthesized de novo by the scutellum and aleurone layer and others are found preformed in the starchy endosperm. The amylases are the best known of these types of enzymes. α-Amylases are synthesized and secreted by the scutellum and aleurone layer, and in the case of aleurone isoforms their synthesis is regulated by GAs and ABA.^2,3 Whereas GAs stimulate the synthesis of α-amylases and many other secreted hydrolases, ABA inhibits these processes. β-Amylases, on the other hand, are preformed enzymes whose synthesis is not affected by ABA and GAs. Two forms of β-amylase are found in wheat grains, one is a soluble form present in ungerminated grains which can form high-molecular-weight homopolymers or heteropolymers; the other is bound in an inactive form via S-S linkages to proteins at the periphery of starch grains.^4,5 The activation of β-amylases is thought to result at least in part from their release from endosperm proteins by the action of GA-induced proteases secreted from the aleurone layer.⁶To examine in more detail the effect of H₂S on wheat grain amylases we used de-embryontated wheat grains where only the aleurone layer and starchy endosperm were the possible sources of amylase activity. Embryoless half grains of wheat (Triticum aestivum L., c.v Yangmai 158) were incubated in water, the H₂S donor NaHS, GA, or combinations of these treatments for up to 12 h. After incubation soluble proteins were isolated from grains by homogenizing in buffer and amylase activity was determined colorimetrically using starch-I₂KI. Figure 1A shows that during the first 8 h of incubation there was no increase in amylase activity from half grains incubated in H₂O or GA but there was a small but significant increase in activity after 10 h and 12 h of incubation in these two treatments. By contrast, incubation in NaHS brought about a three-fold increase in amylase activity by six h and this increased to about five-fold above the initial activity by 12 h of incubation.Open in a separate windowFigure 1Amylolytic activity from wheat half grains measured colorimetrically (A), by diffusion into agar-starch (B) and following native gel electrophoresis (C and D). (A) Half grains were incubated in water (CK), 0.5 mM NaHS, 10 µM GA3 and 0.5 mM NaHS plus 10 µM GA3 for up to 12 h. Amylase activity was measured colorimetrically on grain homogenates by the starch I-KI method. (B) Half grains pre-incubated as (A) then transferred to agar containing 0.2% starch and 2 mM EDT A for a further 12 h. Amylolytic activity was determined by the diameter of the starch-free halo after flooding the agar-starch plate with I-KI solution. Seeds treated with CK, NaHS, GA₃ and NaHS plus GA₃ were lined from left to right in each plate, respectively. (C and D) Wheat grains incubated for 12 h in H₂O, NaHS, GA₃ and Hb (0.1 g/L) and combinations of these treatments, were homogenized and aliquots of extracts were electrophoresed by non-denaturing PAGE. After electrophoesis gels were soaked in starch, washed and stained with I-KI. Amylolytic activity is shown by cleared areas in the gel. For the gel in (C), homogentes were incubated with 25 mM EDT A to inactivate α-amylase and for (D), they were heated at 70°C to inactivate β-amylase.We confirmed these results by incubating wheat half gains on 0.2% agar containing 0.2% soluble starch and 2 mM EDTA (Fig. 1B). In this experiment, half grains were first incubated in H₂O, GA or NAHS for up to 12 h as for the experiment shown in Figure 1A and half grains were transferred to agar to estimate amylase activity. Because α-amylases are Ca²⁺-containing metalloenzymes whose activities are dependent on Ca²⁺ binding we incorporated EDTA into the agar to favor the appearance of β-amylase activity. The data in Figure 1B confirm what we observed when we measured amylase activity colorimetrically, namely that starch degrading activity was high in half grains exposed to NaHS but low in those incubated in H₂O or GA. From this experiment we also concluded that the starch degrading activity was likely a result of the activity of β-amylase as α-amylase activity would have been reduced or eliminated by the presence of EDTA.We established that the amylolytic activity produced by wheat grains in response to the NaHS donor was largely β-amylase by selectively inhibiting amylase activities following native gel electrophoresis. α-Amylases are heat stable but are sensitive to metal chelators, whereas β-amylases are denatured by heating but are not inhibited by chelators such as EDTA.⁴ Figure 1C and D show the activities of amylase isoforms measured by incubating non-denaturing polyacrylamide gels in 1% soluble starch followed by staining in I-KI. Figure 1C shows amylolytic activity after incubation of the gel in starch containing 25 mM EDTA. Two distinct sets of bands are seen together with minor bands of activity. Whereas group I amylases do not change significantly following treatment incubation in the presence of NaHS or GA, group II amylases show high activity in the presence of NaHS, but show no activity with GA (Fig. 1C). When the enzyme preparations were heated to 70°C for 15 min before electrophoresis almost all activity was lost showing that the activity seen in the absence of heating but in the presence of EDTA was likely that of β-amylase. We also used hemoglobin (Hb) a nonspecific H₂S scavenger to show that the effects of NaHS were indeed via the production of H₂S. Figure 1C shows that Hb almost completely abolished the inductive effect of NaHS on β-amylase activity.Although at present we have no direct evidence that H₂S acts as an endogenous regulator of endosperm function in cereal grains, it is tempting to speculate that this is indeed the case. Our previous work showed an increase in the synthesis of H₂S by wheat grains that is detected as early as 12 h following the start of imbibition in H₂O.¹ Although we have no information on the route of H₂S synthesis in wheat, there is abundant evidence that plants synthesize H₂S as its emission has been observed in many species.^7–13 In plants, H₂S is thought to be released from cysteine via a reversible O-acetylserine (thiol) lyase (OASTL) reaction, and recently several L- and D-cysteine-specific desulfhydrase candidates have been isolated and partially characterized from Arabidopsis thaliana, confirming H₂S release by the action of desulfhydrases in various cellular compartments.^14–17 The induction of L-cysteine desulfhydrase upon pathogen attack,¹⁸ freezing tolerance by H₂S fumigation,¹⁹ emission of H₂S from plants exposed to SO₂ injury,^10,20 and abiotic stresses tolerance in plants treated with H₂S donor,^1,21–24 all infer that H₂S is involved in these responses.It is also tempting to speculate that H₂S may work in cereal grains by influencing redox status.^15,25 It has been proposed that H₂S can bring about an increase in synthesis of glutathione from cysteine and an overall improvement of plant performance under stress. The release of β-amylase from starchy endosperm proteins or the dissociation of free β-amylase from small homopolymers or heteropolymers has been shown to be enhanced by reducing agents such as dithiothreitol and by S-H proteinases. We propose that one plausible action of H₂S in cereal endosperm is to enhance reactive sulfur species that can lead to reduction of S-S bonds between β-amylase and its binding partners in the endosperm. Release of activated β-amylase would then be free to participate in starch degradation providing sugar units for seedling growth and development prior to the induction of α-amylases by GAs.     

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

Crystal toxin Cry1Ca from Bacillus thuringiensis has an insecticidal spectrum encompassing lepidopteran insects that are tolerant to current commercially used B. thuringiensis crops (Bt crops) expressing Cry1A toxins and may be useful as a potential bioinsecticide. The mode of action of Cry1A is fairly well understood. However, whether Cry1Ca interacts with the same receptor proteins as Cry1A remains unproven. In the present paper, we first cloned a cadherin-like gene, SeCad1b, from Spodoptera exigua (relatively susceptible to Cry1Ca). SeCad1b was highly expressed in the larval gut but scarcely detected in fat body, Malpighian tubules, and remaining carcass. Second, we bacterially expressed truncated cadherin rSeCad1bp and its interspecific homologue rHaBtRp from Helicoverpa armigera (more sensitive to Cry1Ac) containing the putative toxin-binding regions. Competitive binding assays showed that both Cry1Ca and Cry1Ac could bind to rSeCad1bp and rHaBtRp, and they did not compete with each other. Third, Cry1Ca ingestion killed larvae and decreased the weight of surviving larvae. Dietary introduction of SeCad1b double-stranded RNA (dsRNA) reduced approximately 80% of the target mRNA and partially alleviated the negative effect of Cry1Ca on larval survival and growth. Lastly, rSeCad1bp and rHaBtRp differentially enhanced the negative effects of Cry1Ca and Cry1Ac on the larval mortalities and growth of S. exigua and H. armigera. Thus, we provide the first lines of evidence to suggest that SeCad1b from S. exigua is a functional receptor of Cry1Ca.     

Identification and expression analysis of four heat shock protein genes associated with thermal stress in rice weevil, Sitophilus oryzae

In order to explore the function of heat shock proteins during thermal stress in rice weevil, Sitophilus oryzae, four heat shock protein genes were cloned and characterized. These heat shock protein genes (hsps) were named as Sohsp70–1, Sohsp70–2, Sohsc70, and Sohsp90, respectively. These hsps showed high sequence conservation with the maximum identity with hsps of Tribolium castaneum and other insects. All the four genes showed the highest mRNA expression in pupal stage and the lowest levels in larval stage. The induced expression of the two Sohsp70s (Sohsp70–1 and Sohsp70–2) were reached to the highest levels (15.59-fold and 12.66-fold) after 2?h of incubation at 37?°C, respectively. Expression of Sohsp90 not only was significantly elevated by heat stress but also by cold stress. Whereas, expression level of Sohsc70 was not induced either by heat or cold stress. Furthermore, for rapid heat hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were observed as 2.57, 2.53, 3.33 and 2.33-fold higher than control, respectively; for rapid cold hardening, the expression levels of Sohsp70–1, Sohsp70–2, Sohsc70 and Sohsp90 were reported as 2.27, 3.02, 3.37 and 2.23-fold higher than control, respectively. Hence, our results revealed that the four Sohsps were associated with temperature adaption under rapid heat or cold hardening.     

泥炭地特征性环境因子促进泥炭藓持久孢子库的形成

研究泥炭地特征性环境因子——淹水、少氧和化感物质对泥炭藓孢子持久性的影响, 可深入理解泥炭地泥炭藓持久孢子库的形成机制, 为退化泥炭地泥炭藓地被恢复研究提供参考。该研究以藓丘种和丘间种两种泥炭藓的孢子为试验材料, 通过室内模拟控制实验的方法, 研究泥炭藓孢子在空气、超纯水、泥炭地地表水和泥炭藓沥出液中, 及3种速率充气下, 孢子萌发力持久性的变化。经充气处理后, 泥炭藓孢子持久性显著低于不充气处理。不充气时, 泥炭藓孢子在含有化感物质的泥炭地地表水和泥炭藓沥出液中保存, 持久性显著高于在超纯水中保存。通径分析结果显示, 溶解氧是影响泥炭地泥炭藓孢子持久性的主要因子和限制因子, 养分元素氮(TN)和磷(TP)的浓度为孢子持久性的负作用因子。研究结果表明, 泥炭藓孢子散布于苔藓地被基质或淹水的丘间生境中, 比暴露于空气或在无化感物质的水中, 能更好地维持萌发力。泥炭地中, 泥炭藓孢子和其他植物的繁殖体的超长寿命可能归因于少氧、养分贫乏和丰富的化感物质等泥炭地特征性环境因子。