虫生真菌分子致病机理及基因工程改造研究进展

虫生真菌侵染寄主昆虫的复杂过程可分为体表附着、体壁穿透及体内定殖和致死等不同阶段.近年来,以金龟子绿僵茵(Metarhizium anisopliae)和球孢白僵菌(Beauveria bassiana)为代表的基因功能研究取得了长足的进展,从不同角度阐明了虫生真菌的分子致病机理;同时,基因工程技术的应用为昆虫病原真菌的遗传改良和选育高毒力杀虫菌株开辟了新的途径.对近年来昆虫病原真菌侵染寄主的分子对策及基因工程改造的研究进展进行了综述,并就进一步研究虫生真菌的毒力基因及功能进行了探讨.     

昆虫病原真菌基因研究进展*

昆虫病原真菌是一类重要的害虫自然控制因子,但由于病原真菌对寄主昆虫的慢击倒性,使得病原真菌的应用受到极大的限制。八十年代,人们的研究目标集中在昆虫病原真菌的酶学研究上;到了九十年代则全面进入了分子生物学的研究阶段。包括昆虫病原真菌毒力基因在内的许多基因得到了研究,使得对病原真菌的人工定向改造成为可能。1 蛋白酶基因昆虫的体壁主要是由蛋白基质和分布其中的几丁质组成,是防止大多数微生物入侵的有效途径。蛋白质的类型和数量在不同种类的昆虫、组织和生长阶段中是不断变化的（Andersen,1974）。为了能穿透这样一…     

虫生真菌分子生物学研究进展*

虫生真菌分子生物学的在研究自开展以来,有20多种与杀虫毒力相关的蛋白酶得到纯化,而与之相对应的基因也分别得到克隆、测序。陆续的基因克隆表明,虫生具菌中的重要毒力基因以基因族的形式存在,在精确的表达调控下以适应不同的寄主种类和不同的环境条件。基因工程研究表明,以增加毒力基因拷贝的方式可显提高虫生真菌的杀虫速率,带有外源基因标记的菌株也是环境释放示综研究的良好材料及方法。     

根虫瘟霉转寄主过程中毒力相关胞外蛋白酶系的诱导表达

根虫瘟霉Zoophthoraradicans是寄主范围较广的专性昆虫病原真菌。在该菌胞外蛋白酶系与毒力变化关系的研究中,不同寄主来源的根虫瘟霉菌株产胞外蛋白酶水平与其对小菜蛾(Plutellaxylostella)的毒力间无明显的相关性。但转寄主各代菌株随毒力逐步上升产胞外蛋白酶水平也有所增加,两者间有一定的相关性。经活性电泳检测显示,ARSEF1342原始菌株(R0)有分子量分别为148kD,153kD和162kD的三条蛋白酶条带,但转寄主传代菌株(R1~R4)的148kD蛋白酶条带突然消失,而153kD蛋白酶条带则随转寄主传代数增加逐步趋于明显,相关性分析发现153kD和148kD与毒力间具有较好的相关性。这表明根虫瘟霉菌株在转寄主过程中逐渐增加了对新寄主具有较高基质特异性的胞外蛋白酶的诱导表达,从而使转寄主菌株更适应对新寄主的入侵及致病。     

马尾松林中球孢白僵菌寄主转移和专化性的SSR标记分析

球孢白僵菌是一种最常见的虫生真菌,已被广泛应用于害虫生物防治。利用SSR简单序列重复(simple sequence repeat,SSR)分子标记对来自安徽省麻姑山马尾松林的102株球孢白僵菌进行基因分型、寄主转移和寄主专化性研究。9对微卫星引物将102株白僵菌分成31个微卫星基因型。在这31个微卫星基因型中,有5种基因型株系为相对优势菌株,这5种基因型株系通过侵染不同寄主昆虫即寄主转移延续自身在马尾松林生态系统中的传播和流行,从而证实寄主转移是白僵菌群体中的普遍现象。同时,这些相对优势基因型并不是在各个月份均匀分布,在大部分月份中,存在1–2种优势度高的基因型。相同基因型株系可侵染不同寄主的结果揭示出白僵菌不仅在种的水平,而且在菌株水平上寄主专化性也较弱。正是白僵菌较弱的寄主专化性特征促使部分基因型株系通过寄主转移,在马尾松林生态系统中得以宿存和延续。     

基因工程改良在昆虫病原真菌中的应用

昆虫病原真菌是自然界控制害虫种群的主要生物因子,其研究开发越来越受到重视。但由于存在致死速度慢、防效不稳定、对环境条件要求高等缺点,限制了昆虫病原真菌的应用。近年来,通过基因工程技术对昆虫病原真菌进行遗传改造,提高菌株的致病性和毒力,创造高效、稳定的工程菌株取得了较大进展。对昆虫病原真菌选择标记、遗传转化方法、基因工程改良及应用等方面最新研究进展进行了综述。     

Virulence of Metarhizium flavoviride 82 to different developmental stages of the brown planthopper, Nilaparvata lugens (Hemiptera:Delphacidae)

对水稻重要害虫褐飞虱Nilaparvata lugens目前市场上尚无一种理想的微生物杀虫剂.昆虫病原真菌具有从体壁侵入的能力因而对刺吸性害虫的防治具有优势.为此,本研究选用不同原寄主和来源地的3种昆虫病原真菌(金龟子绿僵菌Metarhizium anisopliae、黄绿绿僵菌Metarhizium flavoviride和球孢白僵菌Beauveria bassiana)的12个不同菌株,以1 100孢子/mm2孢子悬浮液进行室内毒力测定.结果表明:在参试的不同菌种12个菌株中,黄绿绿僵菌Mf82菌株对褐飞虱成虫致病力最高,10 d累计校正死亡率为83.5％,致死中时(LT50)为4.6d.其不同浓度孢子液对褐飞虱3个发育阶段有不同程度的致病力,毒力大小顺序为成虫＞高龄若虫＞低龄若虫.黄绿绿僵菌孢子液对各处理稻株褐飞虱产卵痕部位、卵粒均有侵染作用,10d侵染率分别为66.7％和51.2％,卵龄越低,侵染效果越好,卵龄为0.5d时侵染率最高.本研究表明黄绿绿僵菌Mf82菌株对褐飞虱成虫、若虫和卵均有较强的致病性,是一株极具应用潜力的生防真菌.     

昆虫病原线虫感染寄主行为研究进展

昆虫病原线虫斯氏属Steinernema和异小杆属Heterorhabditids线虫是新型的生物杀虫剂。其感染期幼虫是惟一能够侵染寄主昆虫的虫态。这类线虫感染寄主的行为分为寻找寄主、识别寄主和侵染寄主。文章综述昆虫病原线虫感染寄主昆虫的行为以及在感染寄主过程中的影响因素。     

粉棒束孢Isaria farinosa的研发进展

粉棒束孢Isaria farinosa是一种常见的昆虫病原真菌,已被用作一种生物因子防治温带农林害虫、植物病原和线虫。同时,粉棒束孢作为冬虫夏草中的定殖真菌,严重危害冬虫夏草菌Ophiocordyceps sinensis寄主蝙蝠蛾幼虫的饲养,引起了资源昆虫研究者的高度重视和控制。作为生防真菌,粉棒束孢资源的分布和筛选、分子鉴定和遗传多样性、培养、活性成分和药效、分子生物学、害虫生物防治、病害控制、安全性等是学术界的研究重点;作为冬虫夏草寄主昆虫病原菌,学界和产业界一直研究其感染特征、侵染机理和防霉剂筛选等,为这一真菌的高效安全控制提供基础。本文将综述粉棒束孢在利用和控制两大方面的研发进展。     

蚧虫的病原真菌及其在生物防治中的潜力

蚧虫（半翅目：蚧总科）是农林果树和花卉的一类重要害虫。作者综述了寄生蚧虫的虫生真菌及其在生物防治中的潜力。总结了昆虫病原真菌作为生物杀虫剂的研究历史,并将其划分为3个发展阶段,即开创阶段、缓慢发展阶段和快速发展阶段。讨论了该领域在中国的研究现状。列出了世界上目前已记录的蚧虫病原真菌,包括55属140种,及其寄主蚧虫的名录。对虫生真菌未来的研究和开发提出了4点建议。     

Permissive Replication of Homologous Murine Rotavirus in the Mouse Intestine Is Primarily Regulated by VP4 and NSP1

Homologous rotaviruses (RV) are, in general, more virulent and replicate more efficiently than heterologous RV in the intestine of the homologous host. The genetic basis for RV host range restriction is not fully understood and is likely to be multigenic. In previous studies, RV genes encoding VP3, VP4, VP7, nonstructural protein 1 (NSP1), and NSP4 have all been implicated in strain- and host species-specific infection. These studies used different RV strains, variable measurements of host range, and different animal hosts, and no clear consensus on the host range restriction determinants emerged. We used a murine model to demonstrate that enteric replication of murine RV EW is 1,000- to 10,000-fold greater than that of a simian rotavirus (RRV) in suckling mice. Intestinal replication of a series of EW × RRV reassortants was used to identify several RV genes that influenced RV replication in the intestine. The role of VP4 (encoded by gene 4) in enteric infection was strain specific. RRV VP4 reduced murine RV infectivity only slightly; however, a reassortant expressing VP4 from a bovine RV strain (UK) severely restricted intestinal replication in the suckling mice. The homologous murine EW NSP1 (encoded by gene 5) was necessary but not sufficient for promoting efficient enteric growth. Efficient enteric replication required a constellation of murine genes encoding VP3, NSP2, and NSP3 along with NSP1.     

Global host immune response: pathogenesis and transcriptional profiling of type A influenza viruses expressing the hemagglutinin and neuraminidase genes from the 1918 pandemic virus

To understand more fully the molecular events associated with highly virulent or attenuated influenza virus infections, we have studied the effects of expression of the 1918 hemagglutinin (HA) and neuraminidase (NA) genes during viral infection in mice under biosafety level 3 (agricultural) conditions. Using histopathology and cDNA microarrays, we examined the consequences of expression of the HA and NA genes of the 1918 pandemic virus in a recombinant influenza A/WSN/33 virus compared to parental A/WSN/33 virus and to an attenuated virus expressing the HA and NA genes from A/New Caledonia/20/99. The 1918 HA/NA:WSN and WSN recombinant viruses were highly lethal for mice and displayed severe lung pathology in comparison to the nonlethal New Caledonia HA/NA:WSN recombinant virus. Expression microarray analysis performed on lung tissues isolated from the infected animals showed activation of many genes involved in the inflammatory response, including cytokine, apoptosis, and lymphocyte genes that were common to all three infection groups. However, consistent with the histopathology studies, the WSN and 1918 HA/NA:WSN recombinant viruses showed increased up-regulation of genes associated with activated T cells and macrophages, as well as genes involved in apoptosis, tissue injury, and oxidative damage that were not observed in the New Caledonia HA/NA:WSN recombinant virus-infected mice. These studies document clear differences in gene expression profiles that were correlated with pulmonary disease pathology induced by virulent and attenuated influenza virus infections.     

A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.     

粳稻子预44抗LP11稻瘟病菌基因Pizy6(t)的定位

稻瘟病是世界范围内严重威胁水稻(Oryza sativa)生产可持续发展的主要病害之一,每年造成10%–30%的水稻产量损失。抗瘟水稻品种的培育和育种利用是解决稻瘟病危害最经济有效的方法。对新的致病性菌株进行分离和筛选是定位与克隆抗病新基因及抗病育种的基础。选择分离自不同稻瘟病发生重灾区的单孢菌株,对广谱抗瘟水稻子预44和感病水稻江南香糯进行致病性鉴定,筛选出两材料间致病性差异明显的5个菌株;进一步利用子预44、湘资3150、9311、日本晴、丽江新团黑谷、中花11、TP309和江南香糯8个抗瘟性不同的水稻材料,对筛选的菌株进行致病性鉴定。结果显示,LP11能使广谱抗瘟籼稻湘资3150严重发病,推测其很可能是新进化出来的强致病菌株。利用子预44和江南香糯杂交构建的F2群体进行抗性遗传分析,结果表明子预44对LP11菌株的抗性是由单显性基因控制。利用SSR分子标记和图位克隆方法在子预44中定位了1个抗稻瘟病基因Pizy6(t)。研究结果不仅为抗病相关研究提供了有价值的新菌株,而且为子预44中抗稻瘟病基因Pizy6(t)的克隆奠定了基础。