“高效灭蚊幼转基因蓝藻技术的研究”通过鉴定

由山东省寄生虫病防治研究所承担的山东省自然科学金项目——“高效灭蚊幼转基因蓝藻技术的研究”,1992年9月23日在济南由山东省教委、山东省卫生厅及山东省医科院联合组织召开鉴定会,通过了技术鉴定。该项研究是利用基因工程手段将球形芽抱杆菌毒蛋白基因转移入蓝藻细胞进行表达,结果得到一种易被蚊幼取食的高效灭蚊幼蓝藻。转基因蓝藻完整细胞对淡色库蚊半致死浓度(24h)为1.9～5.0×10~4个/ml,2.0×10~6个/ml 浓度时24h 即可杀死全部测试幼虫,对中华按蚊等幼虫也有显著毒效。参加鉴定的专家们一致认为,该研究在国际蚊虫生物防制的热点问题上取得较大进展;利用遗传工程手段在藻类应用领域寻找到了一个新的方向;其中某些结果也在蓝藻分子遗传学领域具有一定价值。这项研究为国内首创,并达到同类研究的国际领先水平。     

3种基因工程藻对中华按蚊幼虫杀灭作用的实验研究

本实验观察了３种基因工程杀蚊幼蓝藻对中华按蚊Ⅲ龄幼虫的杀灭作用。结果表明，当喷施工程藻浓度高于３．０×１０５ｃｅｌｓ／ｍｌ时，作用２４ｈ后，中华按蚊幼虫死亡数较低，而在４８ｈ、７２ｈ后蚊幼虫大部分（６７％以上）死亡；在喷施工程藻浓度低于３．０×１０４ｃｅｌｓ／ｍｌ时，２４ｈ、４８ｈ和７２ｈ的杀蚊幼效果均较差，此结果明显低于工程藻对淡色库蚊幼虫的作用效果。因此在应用基因工程藻进行现场控制蚊幼虫时，特别是在有较多按蚊幼虫的孳生地，应该考虑联合应用其它防治方法。     

三种革兰阴性细菌在按蚊饲养水体及按蚊中肠内存活能力的研究

目的 探讨分离自中华按蚊雌成蚊中肠的3种革兰阴性细菌,即气单孢菌、A4菌(未鉴定)和肠杆菌在按蚊幼虫孳牛水体中及按蚊体内的存活能力. 方法 3种目标细菌在实验室经数量扩增后,添加到饲养按蚊幼虫的水体中,依次收集饲养盆和蛹盒内的水样品,并解剖羽化后吸血雌成蚊,取其中肠制成研磨液;建立水样品和研磨液中细菌的16S rDNA序列克隆文库,并从各样品中分离可培养细菌,比较各样品中的优势菌种和不同处理中可培养细菌的种类组成. 结果 在所有样品中均未检测到与加入的人工培养细菌种类相同的细菌.按蚊幼虫饲养水体和蛹盒水体中的可培养细菌均以嗜水气单孢菌和睾丸酮丛毛单胞菌为主,在少量按蚊的中肠内含有可培养细菌,但均不是目标细菌;而各处理样品中的优势菌均为铜绿假单孢菌. 结论 3种细菌经人工培养后,不能在按蚊幼虫生活水体中建立种群,导致未能在按蚊成蚊中肠内柃测到这些细菌.     

三种革兰阴性细菌在按蚊饲养水体及按蚊中肠内存活能力的研究

目的 探讨分离自中华按蚊雌成蚊中肠的3种革兰阴性细菌,即气单孢菌、A4菌(未鉴定)和肠杆菌在按蚊幼虫孳牛水体中及按蚊体内的存活能力. 方法 3种目标细菌在实验室经数量扩增后,添加到饲养按蚊幼虫的水体中,依次收集饲养盆和蛹盒内的水样品,并解剖羽化后吸血雌成蚊,取其中肠制成研磨液;建立水样品和研磨液中细菌的16S rDNA序列克隆文库,并从各样品中分离可培养细菌,比较各样品中的优势菌种和不同处理中可培养细菌的种类组成. 结果 在所有样品中均未检测到与加入的人工培养细菌种类相同的细菌.按蚊幼虫饲养水体和蛹盒水体中的可培养细菌均以嗜水气单孢菌和睾丸酮丛毛单胞菌为主,在少量按蚊的中肠内含有可培养细菌,但均不是目标细菌;而各处理样品中的优势菌均为铜绿假单孢菌. 结论 3种细菌经人工培养后,不能在按蚊幼虫生活水体中建立种群,导致未能在按蚊成蚊中肠内柃测到这些细菌.     

三种革兰阴性细菌在按蚊饲养水体及按蚊中肠内存活能力的研究

目的 探讨分离自中华按蚊雌成蚊中肠的3种革兰阴性细菌,即气单孢菌、A4菌(未鉴定)和肠杆菌在按蚊幼虫孳牛水体中及按蚊体内的存活能力. 方法 3种目标细菌在实验室经数量扩增后,添加到饲养按蚊幼虫的水体中,依次收集饲养盆和蛹盒内的水样品,并解剖羽化后吸血雌成蚊,取其中肠制成研磨液;建立水样品和研磨液中细菌的16S rDNA序列克隆文库,并从各样品中分离可培养细菌,比较各样品中的优势菌种和不同处理中可培养细菌的种类组成. 结果 在所有样品中均未检测到与加入的人工培养细菌种类相同的细菌.按蚊幼虫饲养水体和蛹盒水体中的可培养细菌均以嗜水气单孢菌和睾丸酮丛毛单胞菌为主,在少量按蚊的中肠内含有可培养细菌,但均不是目标细菌;而各处理样品中的优势菌均为铜绿假单孢菌. 结论 3种细菌经人工培养后,不能在按蚊幼虫生活水体中建立种群,导致未能在按蚊成蚊中肠内柃测到这些细菌.     

蚊类的一种新的寄生虫 Helicosporidium sp.

旋孢子虫(Helicosporidium)系寄生于昆虫血淋巴内的原生动物,Chapman(1967)首次在路易斯安那野外库蚊幼虫的血淋巴内发现这类原虫。最近作者从路易斯安那查尔斯湖畔屠宰场附近的污水沟内捕获的两条黑须库蚊(Culex nigripalpus)幼虫体内找到一种近似于寄生性旋孢子虫(H.parasiticum),而在宿主特异性、致病性和孢子形态学上都有明显区分的孢子虫,作者称之为蚊类的旋孢子虫Helicosporidium sp.本文就后者对数种蚊虫的感染性进行了试验,以测定其作为蚊虫生物防治剂的可能性。     

苏云金杆菌以色列亚种对蚊幼虫体内解毒酶活性的影响

目的 目的 研究苏云金杆菌以色列亚种 （Bacillus thuringrensis var. israelesis, Bti） 对蚊幼虫体内3种解毒酶活性的影响。 方法 方法 采用室内生物测定的方法, 分别测定淡色库蚊和埃及伊蚊幼虫取食Bti后体内谷胱甘肽转移酶、 乙酰胆碱酯酶和羧酸酯酶的活性。 结果 结果 Bti对蚊幼虫体内3种解毒酶活性均产生影响。蚊幼虫在Bti处理后谷胱甘肽转移酶活性显著增强又迅速下降、 后期持续抑制; 羧酸酯酶活性上升后迅速下降并恢复到正常水平; 乙酰胆碱酯酶活性受Bti影响较小, 前期受到抑制后逐步回升至正常水平。3种酶活性与Bti浓度呈正相关。 结论 结论 Bti可显著影响蚊幼体内谷胱甘肽转移酶、 乙酰胆碱酯酶和羧酸酯酶的活性。     

加州稻田内植物和被捕食种类对食蚊鱼捕食选择的影响

食蚊鱼可减少稻田内蚊幼虫数量,但报道不一,这可能与替代性被捕食种类的存在,鱼的捕食者的存在以及食蚊鱼对蚊幼虫食物的影响有关。作者于1984,1985年夏在加州稻田中,研究了水生植物和替代性被捕食种类对食蚊鱼捕食环跗库蚊(Culex tarsalis)幼虫的影响。捕食实验前一月,选择长度在35—45mm     

增加苏云金杆菌以色列变种杀按蚊幼虫效能的漂浮食饵制剂

苏云金杆菌以色列变种B.t.i.粗制粉末的各种制剂如:浓缩液或颗粒剂能有效地防制伊蚊、库蚊和鳞蚊幼虫。按蚊幼虫取食时仅过滤最上层水中的颗粒,过滤率约比库蚊和伊蚊幼虫少10～20倍。结果降低了胃毒素B.t.i.的效能。     

食蚊罗索虫体外培养的研究进展

食蚊罗索虫(Romanomermis culicivorax Ross and Smith,1976)是一种专性寄生于蚊幼虫体内的索科线虫。自本世纪60年代在美国路易斯安那等地发现该线虫以来,经过大量的实验室和现场研究,证明是一种颇有前途的生物防制剂。1984年被世界卫生组织列为近期开发对象之一。但迄今为止,食蚊罗索虫的培养还只能采用体内培养的方法,即用寄生前期幼虫实验室感染蚊宿主幼虫,获得寄生后期幼虫。这种方法虽然不算复杂,但必需饲养蚊虫,需要     

Invasion of toxic marine cyanobacteria in to the tsunami affected coastal villages of southern India

This documentation explores the facts about the invasion of marine cyanobacteria in to the tsunami affected coastal villages of Nagapattinam district of Tamilnadu and Karaikkal district of Pondicherry Union Territory (UT) in southern India. Water samples were collected from eight tsunami-hit coastal villages in different open water sources. The collected samples were processed for detecting marine cyanobacterial growth. Totally 110 water samples were processed, three samples were positive for the toxic cyanobacteria, Lyngbya sp., and nine for nontoxic species such as Epithemia sp.,, Johannesbaptistia pellucida, Oscillatoria princeps, Phormidium fragile, Synechocystis sp. Besides posing a public health risk because of the toxic cyanobacteria, the bloom formation by the cyanobacterial species such as Anabaena, Microcystis, Lyngbya, Plectonema, Phormidium contaminated the water bodies and deteriorated the water quality in the tsunami affected villages. The study revealed that another kind of public health risk from the invasion of toxic cyanobacteria to the costal ecosystem during the tsunami. It is necessary, in this context, that the surveillance mechanism, which is geared up during or after natural disasters, should have a provision to monitor the transportation of toxic elements/organisms from marine system to coastal/inland ecosystems and to control such organisms.     

Investigations of the immunomodulatory effect of cyanobacterial extracts]

Resulting from the knowledge that cyanobacteria (blue-green algae) are able to produce pharmacologically active substances the aqueous extracts from several cyanobacteria species and strains (Microcystis aeruginosa, Synechocystis aquatilis, Oscillatoria redekei, Anabaena flos-aque, Aphanizomenon flos-aquae, Oscillatoria rubescens, Oscillatoria tenuis) were tested for their immunomodulating activity. Extracts from Oscillatoria redekei 051, Oscillatoria tenuis 01 and Synechocystis aquatilis 428 caused an immunosuppression. They inhibited not only the incorporation of 3H-thymidine into mitogen stimulated lymphocytes but reduced also the number of plaque-forming cells of mice as shown by hemolysis-plaque-assay. Only extracts from Oscillatoria redekei 051 did not show any cytotoxic effects in lymphocyte cytotoxic test. This may be an evidence for a specific action on the proliferation of lymphocytes.     

Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120

Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.     

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.     

Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120

In response to combined nitrogen starvation in the growth medium, the filamentous cyanobacterium Anabaena sp. PCC 7120 is able to develop a particular cell type, called a heterocyst, specialized in molecular nitrogen fixation. Heterocysts are regularly intercalated among vegetative cells and represent 5-10% of all cells along each filament. In unicellular cyanobacteria, the key Krebs cycle intermediate, 2-oxoglutarate (2-OG), has been suggested as a nitrogen status signal, but in vivo evidence is still lacking. In this study we show that nitrogen starvation causes 2-OG to accumulate transiently within cells of Anabaena PCC 7120, reaching a maximal intracellular concentration of approximately 0.1 mM 1 h after combined nitrogen starvation. A nonmetabolizable fluorinated 2-OG derivative, 2,2-difluoropentanedioic acid (DFPA), was synthesized and used to demonstrate the signaling function of 2-OG in vivo. DFPA is shown to be a structural analogue of 2-OG and the process of its uptake and accumulation in vivo can be followed by (19)F magic angle spinning NMR because of the presence of the fluorine atom and its chemical stability. DFPA at a threshold concentration of 0.3 mM triggers heterocyst differentiation under repressing conditions. The multidisciplinary approaches using synthetic fluorinated analogues, magic angle spinning NMR for their analysis in vivo, and techniques of molecular biology provide a powerful means to identify the nature of the signals that remain unknown or poorly defined in many signaling pathways.     

PCR及PCR-ELISA法检测蚊体内马来丝虫幼虫

目的：建立一种检测蚊体内马来丝虫幼虫的灵敏、快速、特异的方法。方法：选择应用PCR及PCR－ELISA法检测马来丝虫幼虫DNA的最佳反应条件，并在该条件下分别以马来丝虫Ⅰ期、Ⅱ期、Ⅲ期幼虫各1条作模板，测定检测的灵敏度及检测实验室人工感染中华按蚊体内的马来丝虫幼虫。结果：PCR及PCR－ELISA法均能检测出1条Ⅰ期幼虫（L1），而PCR的检测下限为1／10条L1，PCR－ELISA检测下限为1／100条L1；将分离的感染期幼虫加阴性蚊媒进行粗提、扩增及电泳，结果未见明显的扩增条带，扩增产物作ELISA检测，全部为阴 性；个体解剖人工感染的中华按蚊120只，分别收集113只阳性蚊体内的幼丝虫，用两种方法检测，结果均为阳性。结论：初步建立了PCR及PCR－ELISA法检测蚊体内马来丝虫幼虫的方法。     

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the beta-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the alpha-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.     

A cloned cyanobacterial gene for glutamine synthetase functions in Escherichia coli, but the enzyme is not adenylylated

The coding sequence for Anabaena 7120 glutamine synthetase [L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.1] are shown to be contained within a 7.5-kilobase-pair (kbp) HindIII fragment that has been cloned by plaque hybridization. The hybridization probe for the cyanobacterial gene was a recombinant plasmid containing the glnA gene from Escherichia coli K-12. Evidence that the cloned Anabaena fragment contains the glnA gene includes complementation of a glnA deletion mutant of E. coli and immunological identity of the enzyme produced by the cloned Anabaena fragment in E. coli with glutamine synthetase purified from Anabaena 7120. Heteroduplex analysis reveals 0.65 kbp of homology between the 7.5-kbp Anabaena 7120 fragment and an 11-kbp E. coli fragment that codes for E. coli glutamine synthetase. Studies of Anabaena glnA gene activity in E. coli suggest that the cyanobacterial gene is not repressible and that the Anabaena 7120 glutamine synthetase is not adenylylated in E. coli.     

水蚤对球形芽孢杆菌灭蚊幼效果的影响

本文结果证明,在蚊虫孳生地中,与蚊幼共存的水蚤是影响B.S灭蚊效果的极为重要的生态限制因子,它可使B.S对蚊幼虫的杀灭率下降30％～50％,使B.S持效明显缩短或消失。细菌学观察证实,水蚤对B.S具有快速的清除作用,B.S暴露于水蚤的生境中,于12hB.S菌数下降86％。这一生态限制因子给B.S的杀虫效果带来严重影响,是B.S应用中亟待解决的问题。