串联抗菌肽Cecropin B基因在莱茵衣藻中的表达及其抗菌活性分析

为了应对各种抗生素在水产养殖业所带来的副作用,我们在本文中尝试利用微藻对一种抗菌肽进行表达的可行性研究．根据莱茵衣藻核基因组偏爱密码子对抗菌肽Cecropin B基因进行改造,并将4个经改造的Cecropin B基因依次串联起来,中间加上莱茵衣藻的自剪切连接肽序列LWMRFA,人工合成总长度为522 bp的串联Cecropin B基因．将串联Cecropin B基因克隆到含hsp70-RBCS2启动子和RBCS2终止子的pH105载体上,再与携带ble筛选基因的表达框架连接,构建重组表达载体pCB124．采用玻璃珠转化法将载体pCB124导入莱茵衣藻cc-849中,筛选得到能表达抗菌肽Cecropin B的转基因衣藻．经过6个月的保持培养后,进一步对转基因藻细胞提取液进行抗菌活性分析,发现转基因藻具有明显的抑制革兰氏阴性菌(大肠杆菌)和革兰氏阳性菌(枯草芽孢杆菌和溶壁微球菌)生长的特征．这一结果为具有抗菌活性的饵料藻的生产和应用提供了新的途径．     

抗菌肽在转基因动植物中的研究进展

抗菌肽是一类广泛存在于生物界的小分子短肽,具有广谱的抗菌、抗病毒、抗寄生虫及抗癌等生物活性.将抗菌肽基因转入动、植物体内,不仅可以提高动、植物的抗病能力,而且有望通过转基因动、植物来大量生产抗菌肽,具有重要的研究价值和广阔的应用前景.就抗菌肤的特性、抗菌机理及其在转基因动、植物中的研究做一简要的综述.     

抗菌肽及抗菌肽转基因植物研究进展

抗菌肽是一类小分子多肽,在生物体内分布广泛,具有广谱的抗菌性,是生物体内天然防御系统的一部分。主要介绍了抗菌肽的性质,类型,作用机制及抗菌肽转基因植物的研究进展。     

酵母菌中表达的新疆家蚕抗菌肽（Cecropin-XJ）的特性研究

研究酵母菌中表达的新疆家蚕抗菌肽 (CecropinXJ)的抗菌特性。根据琼脂孔穴扩散法,检测抗菌肽的热稳定性、抑菌效价、对氨苄青霉素抗性菌的抑菌作用,并检测了抗菌肽对酸碱盐、人造胃液的耐受性及其抗菌谱。结果显示新疆家蚕抗菌肽具有很强的热稳定性、能够杀灭氨苄青霉素抗性菌、对酸碱盐、人造胃酸有一定的耐受性,其杀菌活力为：1mg抗菌肽与1200U的氨苄青霉素杀菌活力相当。新疆家蚕抗菌肽能够不同程度地杀灭革兰氏阳性菌和革兰氏阴性菌,这一发现将为抗菌肽在农业、医疗卫生、畜牧业等方面的应用奠定基础,同时为深入研究抗菌肽作用机制提供了依据。     

抗菌肽克隆基因的表达和转基因研究现状

抗菌活性肽具有抗细菌、真菌、病毒、原虫,还有杀精子活性和抑癌活性,具有潜在的应用价值。本文介绍国内外克隆抗菌肽基因的表达研究状况以及抗菌肽在转基因植物、动物方面的应用成果及存在的问题。     

昆虫抗菌肽基因工程研究进展

昆虫抗菌肽对细菌、真菌、病毒和原虫都具有杀灭作用 ,甚至对肿瘤细胞也具有杀伤作用。昆虫抗菌肽并且有独特的作用机制 ,成为众多表达系统外源导入基因的侯选对象之一 ,综述了昆虫抗菌肽的种类及其在微生物转基因工程和植物转基因工程中的进展。     

抗菌肽基因工程研究及其表达策略

抗菌肽广泛存在于多种生物体内,具有广谱抗菌、调节免疫、抑制肿瘤等多种生物学功能,作用机制独特,是目前基因工程研究的热点之一。本文综述了抗菌肽的一般性质及其国内外基因工程研究进展,探讨了在抗菌肽转基因研究中采用的表达策略及理论依据。     

斜纹夜蛾抗菌肽基因Moricin和Cecropin时空表达模式及微生物敏感性的分析

抗菌肽是一类具有广谱抗菌活性的小分子多肽, 包括抗细菌、 病毒和真菌。本研究以斜纹夜蛾Spodoptera litura (Fabricius)为研究对象, 利用Real-time PCR和半定量RT-PCR检测了其体内两个重要抗菌肽基因Cecropin和Moricin的时空表达模式及其对微生物的敏感性。Real-time PCR检测结果表明, 抗菌肽基因Cecropin和Moricin在斜纹夜蛾各个龄期（1, 2, 3, 4, 5和6龄幼虫、 蛹、 成虫）都有表达, 随着龄期的增长, 表达量逐渐增加, 6龄幼虫中表达量达到最高。 半定量RT-PCR检测表明, 抗菌肽基因Cecropin和Moricin在7个不同组织（气管、 卵巢、 马氏管、 体壁、 中肠、 脂肪体和血细胞）中都有表达, Cecropin在脂肪体中表达量最高, 而Moricin在血细胞中表达量相对较高; RT-PCR检测表明, Cecropin和Moricin的表达在大肠杆菌Escherichia coli诱导后迅速上升, 其中诱导后8 h达到高峰期, 一直持续到48 h。不同病原微生物（大肠杆菌E. coli、 金黄色葡萄球菌Staphyloccocus aureus和白僵菌Beauveria bassiana）分别诱导后, Real-time PCR 检测表明, 斜纹夜蛾抗菌肽基因Cecropin和Moricin对大肠杆菌的刺激最为敏感。本研究结果为进一步研究斜纹夜蛾抗菌肽基因Cecropin和Moricin的功能奠定基础。     

抗菌肽研究进展

抗菌肽是广泛存在于自然界生物中的一种具有抗菌、抗病毒和抗肿瘤活性的多态,由于抗菌肽具有普通抗生素所不具有的一系列优点,抗菌肽的研究已经成了现代学术和应用研究的一个热点。作者综述了抗菌肽的抗菌机制和应用研究等方面的内容。     

昆虫抗菌肽基因工程及其分子设计研究进展(英文)

昆虫抗菌肽是昆虫免疫后存在于血淋巴中的一类活性肽,具有广谱抗菌的生物活性,因而昆虫抗菌肽基因工程迅速成为生命科学领域的研究热点之一.介绍了昆虫抗菌肽的基因结构和表达,综述了昆虫抗菌肽的人工合成与改造以及转基因工程的研究成果,概述了昆虫抗菌肽的分子设计方法和现状,并展望了昆虫抗菌肽的应用前景.     

Expression of giant silkmoth cecropin B genes in tobacco

Cecropin B is a small antibacterial peptide from the giant silkmothHyalophora cecropia. To reveal the potential of this peptide for engineering bacterial disease resistance into crops, several cecropin B gene constructs were made either for expression in the cytosol or for secretion. All constructs were cloned in a plant expression vector and introduced in tobacco viaAgrobacterium tumefaciens. A cDNA-derived cecropin B gene construct lacking the amino-terminal signal peptide was poorly expressed in transgenic plants at the mRNA level, whereas plants harbouring a full-length cDNA-derived construct containing the insect signal peptide, showed increased cecropin B-mRNA levels. Highest expression was found in plants harbouring a construct with a plant-gene-derived signal peptide. In none of the transgenic plants could the cecropin B peptide be detected. This is most likely caused by breakdown of the peptide by plant endogenous proteases, since a chemically synthesized cecropin B peptide was degraded within seconds in various plant cell extracts. This degradation could be prevented by the addition of specific protease inhibitors and by boiling the extract prior to adding the peptide. In addition, anionic detergents, in contrast to cationic, zwitter-ionic or non-ionic detergents, could prevent this degradation. Nevertheless, transgenic tobacco plants were evaluated for resistance toPseudomonas solanacearum, the causal agent of bacterial wilt of many crops, andP. syringae pv.tabaci, the causal agent of bacterial wildfire, which are highly susceptible to cecropin Bin vitro. No resistance was found. These experiments indicate that introduction and expression of cecropin B genes in tobacco does not result in detectable cecropin B protein levels and resistance to bacterial infections, most likely due to degradation of the protein by endogenous proteases.     

转Bt基因抗虫水稻的研究进展与生态安全评价

表达杀虫蛋白的转Bt基因植物正在改革着现代农业．综述了国内外转Bt基因水稻及其抗虫性的研究进展及水稻害虫对Bt水稻的抗性风险及抗性管理策略,提出了对转基因Bt水稻进行生态安全风险评价的具体内容．     

Conversion of compatible plant-pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plants

The hrmA gene from Pseudomonas syringae pv. syringae has previously been shown to confer avirulence on the virulent bacterium P. syringae pv. tabaci in all examined tobacco cultivars. We expressed this gene in tobacco plants under the control of the tobacco Delta0. 3 TobRB7 promoter, which is induced upon nematode infection in tobacco roots (Opperman et al. 1994, Science, 263, 221-223). A basal level of hrmA expression in leaves of transgenic plants activated the expression of pathogenesis-related genes, and the transgenic plants exhibited high levels of resistance to multiple pathogens: tobacco vein mottling virus, tobacco etch virus, black shank fungus Phytophthora parasitica, and wild fire bacterium Pseudomonas syringae pv. tabaci. However, the hrmA transgenic plants were not significantly more resistant to root-knot nematodes. Our results suggest a potential use of controlled low-level expression of bacterial avr genes, such as hrmA, in plants to generate broad-spectrum resistance to bacterial, fungal and viral pathogens.     

Transgenic potato plants resistant to the phytopathogenic bacterium Erwinia carotovora

The phytopathogenic bacterium Erwina carotovora spp. which infects potato plants causes severe losses in agriculture. No protective means or resistance traits usable for plant breeding are known. Introduction of a new resistance gene into potato by gene technology leads to a reduced susceptibility of the transgenic plants towards Erwinia carotovora atroseptica infection. Bacteriophage T4 lysozyme is the most active member of a class of bacteriolytic enzymes also detected in several plant species. Secretion of the foreign T4 lysozyme into the intercellular spaces of transgenic potato plants effects a resistance against the phytopathogenic bacterium already at low expression levels.     

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.     

主要医院感染病原菌与社区感染株耐药性比较

目的比较主要医院感染(HAI)病原菌与社区感染(CAI)株的耐药性,指导合理使用抗菌药物。方法收集永康市第一人民医院2003年1月至2006年6月所有标本中分离的主要HAI菌及其CAI株,分别统计其药物敏感试验。采用美国Dade Behring Microscan Walkaway 40全自动细菌鉴定及药敏测试仪及其配套药敏鉴定板测定MIC值。全国医院感染监测网软件和χ2统计分析。结果主要HAI菌为前4种革兰阳性(G )菌依次是金黄色葡萄球菌、表皮葡萄球菌、屎肠球菌、溶血葡萄球菌,前5种革兰阴性(G-)菌依次是大肠埃希菌、肺炎克雷伯菌、鲍曼不动杆菌、铜绿假单胞菌、阴沟肠杆菌。HAI株耐药率普遍高于CAI株,不同的细菌耐药率各具特点。不论HAI株还是CAI株,G 菌对万古霉素的耐药率最低,G-菌对亚胺培南的耐药率均较低,且两者耐药率差异均无显著性。金黄色葡萄球菌、大肠埃希菌、肺炎克雷伯菌、鲍曼不动杆菌4种HAI株对大多数抗菌药物的耐药率明显高于CAI株,其余5种细菌HAI株仅对少数抗菌药物的耐药率高于CAI株。结论HAI菌株耐药性比CAI菌株强,临床应区分感染性质合理使用抗菌药物,有针对性控制感染,从而减少抗菌药物的滥用和细菌耐药性的产生。     

Alterations in SiRNA and MiRNA Expression Profiles Detected by Deep Sequencing of Transgenic Rice with SiRNA-Mediated Viral Resistance

RNA-mediated gene silencing has been demonstrated to serve as a defensive mechanism against viral pathogens by plants. It is known that specifically expressed endogenous siRNAs and miRNAs are involved in the self-defense process during viral infection. However, research has been rarely devoted to the endogenous siRNA and miRNA expression changes under viral infection if the resistance has already been genetically engineered in plants. Aiming to gain a deeper understanding of the RNA-mediated gene silencing defense process in plants, the expression profiles of siRNAs and miRNAs before and after viral infection in both wild type and transgenic anti-Rice stripe virus (RSV) rice plants were examined by small RNA high-throughput sequencing. Our research confirms that the newly generated siRNAs, which are derived from the engineered inverted repeat construct, is the major contributor of the viral resistance in rice. Further analysis suggests the accuracy of siRNA biogenesis might be affected when siRNAs machinery is excessively used in the transgenic plants. In addition, the expression levels of many known miRNAs are dramatically changed due to RSV infection on both wild type and transgenic rice plants, indicating potential function of those miRNAs involved in plant-virus interacting process.     

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.