农杆菌介导的病毒侵染方法在禾本科植物转化上的研究进展

综述了农杆菌介导的病毒侵染方法（Ａｇｒｏｉｎｆｅｃｔｉｏｎ）在禾本科植物转化研究方面的进展．利用农杆菌的Ｔｉ／Ｒｉ质粒载体将病毒或类病毒的核酸导入植物细胞的方法，近年来在禾本科植物包括水稻、大麦、小麦和玉米等具有经济重要性的粮食作物的转化研究上取得了长足的进展．它具有直接、高效、灵敏等特点，在导入植物过程中不需制备病毒或类病毒的核酸，也不需通过介体昆虫的介导．因此，农杆菌介导的病毒侵染方法是人们研究农杆菌及其禾本科寄主的相互关系、病毒的生物学特性、病毒基因功能和核酸序列的很好的方法．     

Genetic engineering of novel genomes of large DNA viruses

Analyses of the function of specific genes and sequences of large DNA viruses such as herpesviruses and poxviruses present special problems because of the size of their genomes (120 to 250 kilobase pairs). Various methods for engineering site-specific insertions or deletions based on the use of selectable markers have been developed and applied for the elucidation of the function of specific DNA sequences, the identification of genes nonessential for virus growth in cell culture, and the expression of foreign genes. These methods should also make possible the construction of viral vectors capable of delivering genes specifying antigens for the prevention of infectious diseases in humans and animals.     

家蚕基因工程载体研究新进展

基因工程分为将外源DNA整合到宿主染色体的种系转移和外源DNA不整合到宿主染色体的瞬时表达,用于家蚕基因工程的载体主要有两类,一类是基于转座子的载体:Minos转座子载体、Mosl元件载体和PiggyBac转座子载体。另一类是基于病毒的载体:杆状病毒载体S、indbis病毒载体和假型反转录病毒载体。家蚕种系转移主要采用piggyBac转座子载体,而外源基因的表达则主要采用杆状病毒载体和家蚕培养细胞组成的NPV-Bm表达系统,Sindbis病毒载体在家蚕RNA干涉的研究中显示出了诱人的前景。     

果树病毒载体研究进展

植物病毒载体作为重要的研究工具被广泛运用于蛋白表达、基因沉默等研究,当前普遍使用的是以烟草花叶病毒载体等为代表的草本植物病毒载体,但其大多不能侵染果树,且稳定性较差,容易丢失插入的外源基因,因此该类载体无法满足果树等多年生植物研究的需要。近年来新兴的果树病毒载体可解决这些难题,为此作者对果树病毒载体的研究进展和发展方向进行综述。当前国内外取得的研究成果主要包括：（1）通过掌握柑橘衰退病毒、柑橘叶斑病毒、李痘病毒、苹果潜隐球形病毒、葡萄病毒A和葡萄卷叶伴随病毒等果树病毒的传播途径、寄主范围、致病力分化、基因功能和表达策略等特性,采用体外转录或农杆菌介导的方式获得了上述果树病毒的全长侵染性克隆。在此基础上,通过在病毒外壳蛋白基因与其邻近的上游基因之间插入外源基因（包括荧光蛋白基因和β-葡萄糖醛酸酶基因等报告基因）,并用该病毒外壳蛋白基因的启动子或其他异源启动子驱动外源基因表达的方式,将上述6种果树病毒的全长侵染性克隆改造成为病毒载体;（2）运用果树病毒载体明确了柑橘衰退病毒、柑橘叶斑病毒、李痘病毒、苹果潜隐球形病毒和葡萄卷叶伴随病毒在植株中的分布、移动规律,及其在细胞中的定位。探寻了柑橘衰退病毒在大翼来檬上产生茎陷点症状的原因,以及交叉保护防治柑橘衰退病的主要机理。果树病毒载体还被作为病毒诱导的基因沉默载体用于基因功能和防病研究;（3）通过选用本地已经存在,且无虫传能力的弱毒株,以及对控制病毒致病和媒介传播能力的基因进行敲除、突变可以解决果树病毒载体研发过程中所遇到的安全风险。由于有些果树病毒仅分布于植株的韧皮部,因此限制了其作为病毒载体在植株中表达外源基因的范围,但由这类果树病毒构建的病毒载体稳定性极高,并且通过添加分泌信号肽基因等方式可以扩大表达产物在植株中的分布和作用范围,因此其在果树病毒研究方面仍然具有很高的应用价值。此外,采用不同病毒来源的异源启动子代替同源重复区来驱动外源基因的表达,可以进一步提高果树病毒载体的稳定性。     

Transgenic plants as tools to study the molecular organization of plant genes

Transgenic plants are generated in nature by Agrobacterium tumefaciens, a pathogen that produces disease through the transfer of some of its own DNA into susceptible plants. The genes are carried on a plasmid. Much has been learned about how the plasmid is transferred, how the plasmid-borne genes are organized, regulated, and expressed, and how the bacteria's pathogenic effects are produced. The A. tumefaciens plasmid has been manipulated for use as a general vector for the transfer of specific segments of foreign DNA of interest (from plants and other sources) into plants; the activities of various genes and their regulation by enhancer and silencer sequences have been assessed. Future uses of the vector (or others like it that have different host ranges) by the agriculture industry are expected to aid in moving into vulnerable plants specific genes that will protect them from such killers as nonselective herbicides, insects, and viruses.     

水稻条纹病毒外壳蛋白自身互作的活性区段研究

水稻条纹病毒(Rice stripe virus,RSV)的外壳蛋白(coat protein,CP)参与病毒转录、复制等多个生物学过程。病毒蛋白之间的互作对病毒侵染活性非常重要,在明确RSV外壳蛋白CP能够自身互作的基础上,通过构建CP蛋白N端、M区段和C端(CP-N、CP-M和CP-C)的酵母表达载体,利用酵母双杂交系统确定外壳蛋白CP自身互作的活性区段。结果表明,N端和C端第1~81个氨基酸是参与水稻条纹病毒CP蛋白自身互作的活性区段。这一研究结果不仅有助于加深理解RSV CP蛋白在病毒基因组稳定、复制过程中的作用,也有助于加深了解RSV、寄主和传播介体之间关系。     

Isolation of mutants of an animal virus in bacteria

Mutants of animal viruses can be isolated in bacteria by recombinant DNA methods. Since no viral functions are required for propagation of recombinants in bacteria, viral mutants with lethal changes in cis- or trans-acting elements can be isolated, as well as partially or conditionally defective mutants. In the cases of viruses with small DNA genomes, such as the tumorigenic simian virus 40 (SV40), the entire viral DNA can be inserted into the bacterial plasmid pBR322 and cloned in Escherichia coli. Recombinant plasmids with a single copy of SV40 DNA cause morphological transformation of mouse cells in culture with the same efficiency as SV40 DNA isolated from virus-infected monkey cells, but the recombinant DNA is noninfectious and replicates poorly in permissive cells. However, SV40 DNA excised from the plasmid replicates as well as authentic viral DNA and is fully infectious. SV40 mutants with small deletions or base substitutions have been isolated by in vitro site-specific or random local mutagenesis of recombinant DNA followed by cloning in E. coli. Many of the mutants thus isolated are defective in specific viral functions.     

植物抗病基因工程研究进展

综合评述了近５年世界各国在植物抗病基因工程方面取得的进展．包括转植物病毒基因组成分（ＣＰ基因、复制酶基因、反义ＲＮＡ及ｓａｔＲＮＡ）、转核酶基因和转植物抗生物质编码基因的抗病毒病植株；转病原细菌解毒基因、转抗菌肽基因和转溶菌酶基因的抗细菌病植株；转几丁质酶基因、转葡聚糖酶基因、转ＲＩＰ基因、转芪合成酶基因的抗真菌病植株等研究所取得的成果．     

番木瓜eIF4E突变基因的构建

真核翻译起始因子4E（eukaryotic translation initiation factor4E,eIF4E）的缺失或突变能影响病毒对植物的侵染,并介导植物对病毒产生抗性。已有研究证明,番木瓜环班病毒的VPg能与番木瓜elF4E（Cp－eIF4E）互作,在此基础上,笔者通过蛋白序列比对和同源建模分析,确定了6个突变位点,采用套叠PCR方法印Cp－eIF4E基因进行点突变,然后,将它们分别连接到酵母双杂交系统和双分子荧光互补系统两套表达载体上,为后续互作实验和抗病功能验证奠定基础。     

A new arbovirus from Aedes albopictus, an Asian mosquito established in the United States

Ten strains of a new arbovirus belonging to the Bunyamwera group (Bunyaviridae) were recovered from field-collected Aedes albopictus mosquitoes in Potosi, Missouri. This evidence indicates that this species may serve as an arbovirus vector in the United States. The urban-suburban distribution, aggressive biting behavior, and broad viral susceptibility of Ae. albopictus may lead to the transmission of viruses of known public health importance and perhaps of viruses hitherto not transmitted to humans because of the feeding pattern of their usual vectors.     

Cloned Cauliflower Mosaic Virus DNA Infects Turnips (Brassica rapa)

Cauliflower mosaic virus DNA cloned in the Sal I site of bacterial plasmid pBR322 infects turnip plants. The cloned viral DNA must be excised from the recombinant plasmid to infect, but need not be circularized and ligated in vitro. The cloned viral DNA lacks site-specific single-strand breaks found in DNA obtained directly from the virus. However, these breaks are reintroduced into the viral genome during multiplication of the virus in the plant host.     

蚜虫的寄主选择与取食行为

本文综述了作为植物病毒主要介体蚜虫的寄主选择和取食行为。蚜虫对寄主的选择主要为有翅蚜。有翅蚜的活动可分为4个行为阶段:停息相、迁飞相,进攻相和定居相。蚜虫的寄主选择主要在迁飞相和进攻相,并按一定的行为序列进行。决定蚜虫寄主选择行为序列执行的因素主要是食物源刺激,蚜虫在活动中若得到适当的正向刺激,就开始取食。由于大多数蚜虫吸食韧皮部,因此对蚜虫的口针导向机制进行了解释。     

Aphids and their transmitted potato viruses: A continuous challenges in potato crops

Aphid is one of the most destructive insect pests on cultivated plants in temperate regions. Their piercing-sucking mouthparts and phloem feeding behavior directly damage crops and deplete plant nutrients. Potato(Solanum tuberosum L.) is one of the most important food sources on the planet, and several aphid species, e.g., Myzus persicae(Sulzer)(green peach aphid) and Macrosiphum euphorbiae(Thomas)(potato aphid)(Hemiptera: Aphididae) colonize potato and transmit several economically important viruses. Aphid-transmitted potato viruses have been emerging all over the world as a very serious problem in potato production, inducing a wide variety of foliar and tuber symptoms, leading to severe yield reduction and loss of tuber quality. In this review, recent advances in understanding the interactions of potato viruses with their hosts, aphid vectors and the environment are described.     

Virus infection of culturable chlorella-like algae and dlevelopment of a plaque assay

Four distinct viruses with double-stranded DNA are known to replicate in Chlorella-like algae symbiotic with hydras and paramecia. An attempt was made to infect a number of cultured Chlorella strains derived from invertebrate hosts with these viruses. One of the viruses, PBCV-1, replicated in two of the algal strains. Restriction endonuclease analysis of the viral DNA showed that the infectious progeny virus was identical to the input virus; thus, Koch's postulates were fulfilled. Viral infection of the two Chlorella strains has allowed the large-scale production of a eukaryotic algal virus and the development of a plaque assay for the virus.     

抗水稻黑条矮缩病转基因材料的鉴定分析

针对水稻黑条矮缩病毒(RBSDV)的核心粒子外壳蛋白基因(S8)、毒质与非结构蛋白基因(S9)及其嵌合基因(S8S9)构建了6个RNAi载体,经农杆菌介导转化武陵粳1号获得转基因植株。通过潮霉素溶液浸泡法对转基因植株进行初步筛选,剔除假阳性植株;再通过PCR从DNA水平进行转基因阳性鉴定;进一步通过q RT–PCR从RNA水平进行转基因表达量分析。选取q RT–PCR表达量高的单拷贝纯系S8后代进行灰飞虱传毒试验,结果表明,含有S8 RNAi片段的转基因水稻材料对RBSDV具有一定抗性。     

RNA-dependent RNA polymerases,viruses, and RNA silencing

Most viruses have RNA genomes that are replicated and transcribed into messenger RNA by viral RNA-dependent RNA polymerases (RdRps), usually in concert with other viral and host factors. Many, if not most, eukaryotes also encode putative RdRps that have been implicated in sequence-specific, RNA-triggered gene silencing. Although the viral and cellular RdRps have no sequence homology, they share functional similarities such as copying messenger RNA templates and intercellular spread of the amplified sequences. Better understanding of viral and host RdRps will improve our ability to control viruses and to use RNA silencing and viruses as tools for research, biotechnology, and medicine.     

Characterization and molecular cloning of a human parvovirus genome

The genome of the small human virus serologically associated with erythrocyte aplasia and erythema infectiosum (fifth disease) is shown to be a linear, nonpermuted, single-stranded DNA molecule with self-priming hairpin termini, properties which are characteristic of the genomes of the family Parvoviridae. This human parvovirus chromosome was molecularly cloned into bacterial plasmid vectors and the cloned DNA was used to explore its relatedness to other mammalian parvovirus serotypes by DNA:DNA hybridization. It is not related to the human adeno-associated viruses but does show a distant evolutionary relationship to genomes of the helper-independent parvoviruses of rodents. This strongly suggests that it is an autonomous parvovirus, and as such is the first example of a member of this group of common animal pathogens to cause disease in man.