植物转基因抗性策略研究进展

通过转基因技术使植物获得抗病性是控制植物病毒感染的一种重要技术途径。RNA干扰技术的出现为植物转基因抗性策略的研究提供了新思路。植物转基因抗性产生方法主要是通过表达不同的病毒蛋白(外壳蛋白、复制蛋白、运动蛋白及其他病毒蛋白)、RNAs(反义RNA、卫星RNA、缺陷干扰RNA、发夹RNA及人工微小RNA)、非病毒基因(核酸酶、抗病毒蛋白抑制子及植物体)、宿主来源的抗性基因(显性抗性基因和隐性抗性基因)及各种宿主防御反应因子等。介绍了以上几种抗性策略并分析了目前尚存在的问题,以期为植物转基因抗性策略研究的学者提供参考。     

植物抗病毒基因及其介导的抗性机理

当前已知的植物抗病毒基因大体上分为5类。第1类为源自病毒的抗病毒基因,它们源于病毒基因组;第2类为与病毒基因相关的其他基因序列,它们或独立存在、或寄生于病毒、或依赖于病毒才能复制,如核酶基因、缺陷干扰颗粒等;第3类为源自植物的抗病毒基因,它们是植物基因的一部分,如病程相关蛋白基因、潜在自杀基因、核糖体失活蛋白基因等;第4类为植物抗体基因,它们是从动物体内获得的具有抵抗植物病毒作用的中和病毒基因;第5类为干扰素基因,它们为控制激素(干扰素)合成的基因,最初在动物体内发现,其后在烟草、番茄、番椒、丁香等植物中也发现干扰素基因。抗病毒基因介导的抗性机理十分复杂,不同基因介导的抗性机理各不相同,同一基因介导的抗性机理也有不同的模型,多数基因介导的抗性机理还未搞清,所以,在抗性机理方面尚未有统一的学说来阐明。今后这一领域的研究应侧重于植物抗病毒基因资源的分类与整理、抗病毒基因介导的抗性分子基础、抗病毒转基因在植物体内表达的影响因子(如转基因重组、沉默等)、新的抗病毒基因的发掘、增强抗病毒转基因生态安全性与遗传稳定性的措施等方面。     

缺失外壳蛋白羧端155-158序列对TMV侵染性的影响

对野生型TMV—U1的外壳蛋白羧端进行缺失突变,观察到外壳蛋白羧端序列缺失4个氨基酸（即外壳蛋白保留154个氨基酸）的TMV154TAG能较强地系统侵染烟草,并高水平表达外壳蛋白,经过二次转接新烟草植株仍保持较强的系统侵染状况,复制完整病毒粒子。结果说明外壳蛋白羧端序列4个氨基酸序列的缺失对烟草花叶病毒感染和复制无严重影响,对利用外壳蛋白羧端缺失型病毒载体表达外源多肽技术具有一定的应用价值。     

Immunodeficiency and clonal growth of target cells induced by helper-free defective retrovirus

The murine acquired immunodeficiency syndrome is induced by a defective retrovirus. To study the role of virus replication in this disease, helper-free stocks of defective Duplan virus were produced. These stocks were highly pathogenic in absence of detectable replicating murine leukemia viruses (MuLVs) other than xenotropic MuLV. They induced expansion of the infected cell population (over 1000-fold), and this cell expansion was oligoclonal in origin and, most likely, arose through cell division. These results suggest that this defective virus is oncogenic, inducing a primary neoplasia associated with an acquired immunodeficiency syndrome as a paraneoplastic syndrome. These data emphasize the need to determine whether virus replication is necessary for the progression of other immunodeficiency diseases, including acquired immunodeficiency syndrome, and whether these diseases also represent paraneoplastic syndromes.     

Interaction of attenuated and virulent tobacco mosaic virus strains

An interfering effect between an attenuated tobacco mosaic virus (TMV) strain (isolated from a tissue culture) and virulent (typical) strain of this virus distinctly manifests itself starting from an interval of 3 days between infection by these strains and reaches a maximum on the fifth day. The protective effect weakens with increasing interval between inoculation with the attenuated and virulent strains.     

Antigenic study of the protein from a defective strain of tobacco mosaic virus

Soluble protein from the defective strain PM2 of tobacco mosaic virus can be recognized as antigenically distinct from the protein of the wild-type tobacco mosaic virus by Ouchterlony and immunoelectrophoretic analyses at temperatures below 37 degrees C. At these temperatures a polymerized state of protein with a characteristic antigenic structure is present in the wild-type virus; this structure is absent from the PM2 preparations. Above 37 degrees C the precipitin patterns for tobacco mosaic virus and PM2 appear identical.     

Nucleotide sequence of the transforming gene of avian myeloblastosis virus

Avian myeloblastosis virus is defective in reproductive capacity, requiring a helper virus to provide the viral proteins essential for synthesis of new infectious virus. This virus arose by recombination of the nondefective helper virus and host cellular sequences present within the normal avian genome. These latter sequences are essential for leukemogenic activity. The complete nucleotide sequence of this region is reported. Within the acquired cellular sequences there is an open reading frame of 795 nucleotides starting with the initiation codon ATG (adenine, thymine, guanine) and terminating with the triplet TAG. This open reading frame could code for the putative transforming protein of 265 amino acids with a molecular weight of approximately 30,000.     

Effect of cocksfoot mottle virus defective RNA on accumulation of virus capsid protein in wheat plants

The functional role of defective cocksfoot mottle virus RNA in regulating the expression of the viral genome was investigated for testing the hypothesis of its possible participation in transactivation of the synthesis of subgenomic RNA encoding the virus coat protein gene.     

Long-term transmission of defective RNA viruses in humans and Aedes mosquitoes

In 2001, dengue virus type 1 (DENV-1) populations in humans and mosquitoes from Myanmar acquired a stop-codon mutation in the surface envelope (E) protein gene. Within a year, this stop-codon strain had spread to all individuals sampled. The presence of truncated E protein species within individual viral populations, along with a general relaxation in selective constraint, indicated that the stop-codon strain represents a defective lineage of DENV-1. We propose that such long-term transmission of defective RNA viruses in nature was achieved through complementation by coinfection of host cells with functional viruses.     

Viral infection across species barriers: reversible alteration of murine sarcoma virus for growth in cat cells

Infection of cat embryo cells by a centrifugally induced aggregate of murine sarcoma virus and feline leukemia virus gave rise to a defective, focus-forming virus which propagated in cat cells, but not in mouse cells. This virus, apparently enveloped with a feline leukemia virus coat, was later subjected to aggregation with murine leukemia virus, whereupon it regained the capacity for growth in mouse cells.     

Nonencapsidated infectious DNA of adeno-satellite virus in cells coinfected with herpesvirus

Adeno-associated satellite viruses produce antigen detectable by immunofluorescence but not infectious virus in tissue culture cells coinfected with herpes simplex virus. Analysis of DNA extracts from these infected cells shows that large amounts of infectious satellite virus DNA are produced but not encapsidated in the system. This result indicates that satellite virus may be defective at the maturation step.     

Titration patterns of a murine sarcoma-leukemia virus complex: evidence for existence of competent sarcoma virions

Stocks of inurine sarcoma virus show titration patterns ranging from one-to two-hit kinetics. The comparison of various titrations of this virus, both with and without added helper virus, to theoretical model systems composed of defined constituents, suggests the existence of a sarcoma virus that does not need coinfectinig murine leukemia virus to be manifested as a focus-forming unit. The behavior of such nondefective particles is compatible with a postulated leukemia-sarcoma virus hybrid.     

春小麦病毒病的鉴定——Ⅰ.电镜观察及病细胞超微结构分析

小麦病叶中观察到大小为50～55nm×330～450nm的病毒粒体,粒体大多为弹状,少数为两端削平的杆状;粒体横切面有3层同心环结构;本病毒粒体主要存在于感病细胞的细胞质中及细胞核的周围;在个别细胞的核质中发现无被膜粒体。细胞质中的病毒粒体常聚集成团粒体结构,此结构的外缘有膜状物包围。未成熟的筛管分子中和胞间连丝附近的病毒粒体呈散乱无规则分布。观察到病毒粒体通过胞间连丝运转的状态及粒体运转对胞间连丝的影响,还发现高尔基体囊胞增生现象和粒体在细胞质中的出芽现象。将电镜观察结果同已报道的有关文献进行比较,认为该病毒为北方禾谷花叶病毒(国内称小麦丛矮病毒)(NCMV)。     

Radiation leukemia virus: quantitative tissue culture assay

Radiation leukemia virus does not propagate in tissue cultures from either Swiss or C57BL mouse embryos, but it does augment focus formation by the defective Moloney leukemia pseudotype of murine sarcoma virus in Swiss mouse cells and thus can be quantitatively assayed.     

Epizootic diarrhea of infant mice: indentification of the etiologic agent

Electron micrographs of intestinal epithelium of infant mice infected with epizootic diarrhea virus have demonstrated intracellular spherical structures measuring 65 to 75 m(micro) in diameter which have a complex morphology resembling several virus particles. They have ben interpreted as being the etiologic agent of this disease. The particles were present in association with, in some cases within, vesicles of the endoplasmic reticulum of intestinal epithelial cells. They were never seen in the nucleus.     

Virus particles and murine leukemia virus complement-fixing antigen in neoplastic and nonneoplastic cell lines

Twenty-seven lines of murine tissue cultures derived from 12 different cell pools and grown on various media were examined with the electron microscope for morphologically detectable virus particles. They were also tested for complement-fixing mouse leukemia virus antigens and for recoverable virus. A 100-percent correlation between results obtained by these two methods is reported.An additional 19 lines from 8 different cell pools were examined for either virus particles or complement-fixing antigens. All lines were assayed for neoplastic transformation. Seven cell pools gave rise to lines showing evidence of contamination with leukemia virus. Since most of these lines had also undergone "spontaneous" neoplastic transformation in vitro, this virus cannot be excluded as a possible cause of the neoplastic change, or of influencing it. The remaining cell pools gave rise to lines with no evidence of contamination with leukemia virus;but most of these lines also underwent similar transformation. These results suggest that "spontaneous" neoplastic transformation can occur in the absence of detectable mouse leukemia virus.     

转录靶向猪Jiv基因shRNA细胞株的建立 及抗猪瘟病毒转基因猪的构建

【目的】构建转入靶向猪Jiv基因shRNA干扰片段的阳性细胞株,通过比较各细胞株对猪瘟病毒增殖的干扰效果,筛选对猪瘟病毒增殖有明显抑制作用的细胞株,为抗猪瘟转基因猪的构建提供材料。【方法】研究设计了靶向猪Jiv基因的4个shRNA干扰片段,并构建插入干扰片段的慢病毒(P1、P2、P3、P4)。将慢病毒分别转染PK-15细胞,阳性细胞接种猪瘟病毒后72 h用实时荧光定量PCR检测猪瘟病毒RNA的量,以比较4种干扰细胞株对猪瘟病毒增殖的干扰效果。将对猪瘟病毒增殖有较好干扰效果的P2慢病毒干扰载体转染猪胎儿成纤维细胞,获得稳定表达靶向猪Jiv基因shRNA干扰片段的猪胎儿成纤维细胞株,作为抗猪瘟病毒转基因猪构建的供体细胞,将细胞核移植到成熟的去核猪卵母细胞中,获得体细胞核移植胚胎,移植入受体母猪输卵管中进行转基因猪构建,对获得的转基因猪进行外源基因的鉴定。【结果】获得了4株转入靶向猪Jiv基因shRNA干扰片段的PK-15细胞株,其中转入P2干扰载体的细胞株对猪瘟病毒的增殖有明显的抑制作用,将转入P2干扰载体的猪胎儿成纤维细胞株为核供体细胞通过体细胞核移植,获得经鉴定为外源基因插入阳性的转基因猪。【结论】细胞Jiv基因的表达对猪瘟病毒的增殖有一定的影响,筛选获得的一个干扰细胞株对猪瘟病毒的增殖有明显的干扰效果;通过体细胞核移植技术获得一头转入靶向猪Jiv基因shRNA干扰片段（P2）的转基因猪。     

Potato spindle tuber virus: a plant virus with properties of a free nucleic acid

Infectious entities, extractable, with phosphate buffer, from tissue infected with potato spindle tuber virus and inciting symptoms on tomato that are typical of this virus, have properties incompatible with those of conventional virus particles. The infectious particles sediment in sucrose density gradients at approximately the same rate as particles with a sedimentation coefficient of 10S, are insensitive to treatment with organic solvents, and can be concentrated by ethanol precipitation. Treatment with phenol changes neither their infectivity nor their sedimentation properties. Infectivity is insensitive to deoxyribonuclease, but at low ionic strength it is sensitive to ribonuclease. At high ionic strength, infectivity partially survives incubation with ribonuclease. These properties, as well as elution patterns from columns of methylated serum albumin, suggest that the extractable infectious agent may be a double-stranded RNA.