外源RNA干涉基因在烟草中的转化及表达

依据RNA干涉机制,以TMV复制酶基因为靶标基因,针对TMV 5个株系复制酶基因间高度同源序列设计引物,经RT-PCR反应获得靶序列,构建靶序列反向重复结构的RNA干涉双元载体.用根癌农杆菌介导将外源基因转化至烟草品种K326基因组中,培育RNA干涉转基因烟草.人工接种病毒验证转基因烟草中外源基因在植物抗病毒能力方面的表达效果,实时荧光定量PCR分析转基因烟草抗病毒能力.结果表明,实验培育的RNA干涉转基因烟草67%对TMV呈现高度抗性;荧光定量PCR分析显示,对TMV具高度抗性的转基因烟草中病毒复制酶基因转录产物mRNA存在很大程度的降解,证实了RNA干涉技术在培育抗病毒烟草品种中的效果.     

TMV复制酶基因靶向的RNA干涉载体构建

以TMV复制酶基因作为RNAi的靶向序列,应用RT－PCR法获得目的DNA序列。依据RNAi机制,以酶切后连接的方法将目的DNA序列正向、反向锚定连接到pUCCRNAi载体质粒,构建含目的序列反向重复结构的RNA干涉中间载体;反向重复结构酶切后插入含超强启动子的pC2300-35s-OCS表达载体,重组的表达载体质粒经冻融法转化到只含辅助质粒的根癌农杆菌中,完成双元载体系统的构建。每步的重组子经特异引物PCR验证和酶切验证有相应的特异条带存在,且测序鉴定序列正确。确认成功构建了TMV复制酶基因靶向的RNAi双元载体,为RNAi技术在植物病毒病害防治中的应用奠定基础。     

烟草花叶病毒和番茄花叶病毒在含N基因烟草上的症状差异是由运动蛋白基因决定的

烟草花叶病毒(TMV)和番茄花叶病毒(ToMV)是烟草花叶病毒属中关系最为密切的病毒, 但它们在含N基因烟草上产生的枯斑大小有明显的差异. 比较了TMV, ToMV及用ToMV运动蛋白基因(MP)精确置换TMV MP后获得的重组病毒T/OMP在不同寄主上的症状差异, 发现T/OMP在含N基因烟草上产生的枯斑大小与ToMV相似. 分析比较TMV, ToMV和T/OMP外壳蛋白和MP在植物体内的积累水平, 发现三者之间没有明显的差异, 而TMV和T/OMP在原生质体中的复制水平也没有差异. 比较TMV, ToMV和T/OMP接种后烟草体内防御相关酶(PAL, POD和PPO)的活性变化, 结果T/OMP和TMV所诱导酶的变化趋势基本一致, 而与ToMV有所差异, 因此认为MP基因功能的差异决定了TMV和ToMV在N基因烟草上枯斑的大小.     

放线菌素D对烟草花叶病毒RNA在离体烟叶中复制的影响

本文报道放线菌素D(AMD)在离体烟叶中对烟草花叶病毒(TMV)的增殖及其核酸(TMV-RNA)复制的影响。按每克叶片用40μgAMD处理,能抑制70％以上叶组织总RNA的合成,在此剂量下AMD对病毒增殖及其核酸复制的影响与用药的时间有密切关系。在接种病毒前5小时或于接种同时给药对病毒增殖和病毒RNA复制都有强烈的抑制作用,可抑制90％以上;而接种后8小时用药就不再表现抑制作用了;接种后24小时用药不但不表现抑制作用,相反对病毒增殖和TMV—RNA的复制都有一定的刺激作用。AMD对TMV增殖和对TMV-RNA复制的影响完全一致。     

烟草花叶病毒和黄瓜花叶病毒双价RNA沉默抗病载体的快速构建*

利用重组PCR技术将烟草花叶病毒(TMV)部分移动蛋白基因（△MP）和黄瓜花叶病毒（CMV）部分复制酶基因（△Rep）连接起来,获得全长约1000 bp的MP-Rep融合基因,将所得融合基因以反向重复的方式与大豆内含子相连,并定向插入到植物表达载体pBIN438上35S启动子下游,构建了含两种不同病毒来源基因的植物表达载体pBIN438-MP-Rep（i/r）。酶切和PCR鉴定证明所构建的载体与预期的设计完全一致。该研究结果为利用RNA沉默原理进行植物广谱抗病研究奠定了基础。     

黄病毒属病毒复制子载体研究进展

RNA复制子是一种能自主复制的RNA载体,保留了病毒非结构蛋白(复制/转录酶)基因,而结构蛋白基因缺失或由外源抗原基因替代,复制/转录酶可控制载体RNA在细胞质中高水平复制以及外源基因的高水平表达。在黄病毒属病毒感染性克隆基础上,其复制子载体得到了成功的构建。黄病毒属病毒复制子为病毒基因组结构功能研究、表达载体构建、假病毒包装及新型疫苗制备等提供了新的技术平台。本文综述黄病毒属病毒复制子的构建原理、方法及应用。     

TMV 54K基因的3个突变体介导抗病性的研究

利用PCR方法分别构建了烟草花叶病毒（TMV）中一个推测为复制酶的54－kD蛋白基因（54K）缺失N端、C端和仅余基因中部261bp的3个缺失突变体,与野生型54K一起克隆入植物中间载体p208,并通过根癌土壤杆菌（Agrobacterium tumefaciens (SDmith et Townsend)Conn）介导的方法转化烟草（Nicotiana tabacum L.）。用TMV侵染转基因植物的R0代和R1代,结果显示这3个缺失突变体均能介导对TMV的抗病性。     

RNA病毒RNA聚合酶的研究进展

自然界仅有ＲＮＡ病毒以ＲＮＡ作为基因载体。依赖于ＲＮＡ的ＲＮＡ聚合酶在这种病毒的增殖复制期起到了非常重要的作用。它一方面以病毒ＲＮＡ为模板复制子代病毒的基因,另一方面也将病毒增殖期间需要的蛋白质和酶类的基因转录成为ｍＲＮＡ,也就是说它担负了复制酶和转...     

用电镜放射自显影术研究TMV-RNA在烟叶细胞中的复制

在放线菌素D(AMD)抑制细胞RNA合成时,用3H-尿嘧啶核苷标记TMV侵染的和健康的烟草叶片,经固定,包埋,切片,RNA酶处理及放射自显影后,在电镜下观察。结果表明,3H-尿嘧啶核苷向病毒基因组或其复制中间体的掺入主要发生在细胞核内,叶绿体内有少量掺入,线粒体内未发现有掺入。因此认为TMV—RNA的合成主要是在缅胞核内进行的。切片如先经蛋白酶和RNA酶处理,再进行放射自显影,则自显影上的银粒几乎全部消失。从超薄切片后的剩余样品取小样,用酚一SDS法和Serva纤维素柱层析,从TMV侵染的烟叶中分离到了抗RNA酶的’H—ds—RNA。从而推测复制中间体在体内很可能主要是单链的,其复制过程很可能不经过双链复制型(RF)。提取到的’H一‘is—RNA：,可能是提取过程中的人为产物。     

TMV和PVY外壳蛋白双基因融合干涉及烟草转化

为了降低烟草花叶病毒(fobacco mosaic virus,TMV)和马铃薯Y病毒(potato virus Y,PVY)复合侵染对烟草带来的危害,本实验找到TMV-CP和PVY-CP基因部分保守序列,将保守序列进行双基因融合,此双基因即为RNAi的靶序列,用限制性内切酶将双基因从pMD18-T载体上切下,正反向连接到pUCCRNAi载体后,经酶切鉴定后定向连接到含超强启动子的pC2300-35S-OCS表达载体上,利用冻融法将此表达载体导入只含辅助质粒的根癌农杆菌中,构建含靶序列反向重复结构的RNAi双元载体系统,提取转化质粒,经酶切验证鉴定表明TMV和PVY外壳蛋白基因植物表达双元载体构建成功.并转化烟草,获得了3株对TMV和PVY抗性显著提高的转基因烟草.     

The Tobacco mosaic virus replicase protein disrupts the localization and function of interacting Aux/IAA proteins

Previously, we identified a correlation between the interaction of the Tobacco mosaic virus (TMV) 126/183-kDa replicase with the auxin response regulator indole acetic acid (IAA)26/PAP1 and the development of disease symptoms. In this study, the TMV replicase protein is shown to colocalize with IAA26 in the cytoplasm and prevent its accumulation within the nucleus. Furthermore, two additional auxin (Aux)/IAA family members, IAA27 and IAA18, were found to interact with the TMV replicase and displayed alterations in their cellular localization or accumulation that corresponded with their ability to interact with the TMV replicase. In contrast, the localization and accumulation of noninteracting Aux/IAA proteins were unaffected by the presence of the viral replicase. To investigate the effects of the replicase interaction on Aux/IAA function, transgenic plants expressing a proteolysis-resistant IAA26-P108L-green fluorescent protein (GFP) protein were created. Transgenic plants accumulating IAA26-P108L-GFP displayed an abnormal developmental phenotype that included severe stunting and leaf epinasty. However, TMV infection blocked the nuclear localization of IAA26-P108L-GFP and attenuated the developmental phenotype displayed by the transgenic plants. Combined, these findings suggest that TMV-induced disease symptoms can be attributed, in part, to the ability of the viral replicase protein to disrupt the localization and subsequent function of interacting Aux/IAA proteins.     

Biological activities of hybrid RNAs generated by 3''-end exchanges between tobacco mosaic and brome mosaic viruses.

Sequences within the conserved, aminoacylatable 3' noncoding regions of brome mosaic virus (BMV) genomic RNAs 1, 2, and 3 direct initiation of negative-strand synthesis by BMV polymerase extracts and, like sequences at the structurally divergent but aminoacylatable 3' end of tobacco mosaic virus (TMV) RNA, are required in cis for RNA replication in vivo. A series of chimeric RNAs in which selected 3' segments were exchanged between the tyrosine-accepting BMV and histidine-accepting TMV RNAs were constructed and their amplification was examined in protoplasts inoculated with or without other BMV and TMV RNAs. TMV derivatives whose 3' noncoding region was replaced by sequences from BMV RNA3 were independently replication competent when the genes for the TMV 130,000-M(r) and 180,000-M(r) replication factors remained intact. TMV replicase can thus utilize the BMV-derived 3' end, though at lower efficiency than the wild-type (wt) TMV 3' end. Providing functional BMV RNA replicase by coinoculation with BMV genomic RNAs 1 and 2 did not improve the amplification of these hybrid genomic RNAs. By contrast, BMV RNA3 derivatives carrying the 3' noncoding region of TMV were not amplified when coinoculated with wt BMV RNA1 and RNA2, wt TMV RNA, or all three. Thus, BMV replicase appeared to be unable to utilize the TMV 3' end, and there was no evidence of intervirus complementation in the replication of any of the hybrid RNAs. In protoplasts coinoculated with BMV RNA1 and RNA2, the nonamplifiable RNA3 derivatives bearing TMV 3' sequences gave rise to diverse new rearranged or recombined RNA species that were amplifiable.     

Interactions between tobacco mosaic virus and the tobacco N gene

The interaction between tobacco mosaic virus (TMV) and tobacco harbouring the N gene is a classical system for studying gene-for-gene interactions in disease resistance. The N gene confers resistance to TMV by mediating defence responses that function to limit viral replication and movement. We isolated the N gene and determined that N belongs to the nucleotide-binding-site-leucine-rich-repeat (NBS-LRR) class of plant disease resistance genes, and encodes both full-length and truncated proteins. Sequence homologies and mutagenesis studies indicated a signalling role for the N protein similar to that seen for proteins involved in defence responses in insects and mammals. The N gene confers resistance to TMV in transgenic tomato, demonstrating the use of the NBS-LRR class of disease resistance genes in engineering crop resistance. From the pathogen side of this interaction, the TMV 126 kDa replicase protein has been implicated as the avirulence factor that triggers N-mediated defence responses. We employed Agrobacterium-mediated expression strategies to demonstrate that expression of the putative helicase region of the replicase protein is sufficient to elicit N-mediated defences. The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this replicase fragment. Thus, both components of this gene-for-gene interaction are now available for studies that address the molecular mechanisms involved in N-mediated TMV resistance.     

Oligomerization and activity of the helicase domain of the tobacco mosaic virus 126- and 183-kilodalton replicase proteins

A protein-protein interaction within the helicase domain of the Tobacco mosaic virus (TMV) 126- and 183-kDa replicase proteins was previously implicated in virus replication (S. Goregaoker, D. Lewandowski, and J. Culver, Virology 282:320-328, 2001). To further characterize the interaction, polypeptides covering the interacting portions of the TMV helicase domain were expressed and purified. Biochemical characterizations demonstrated that the helicase domain polypeptides hydrolyzed ATP and bound both single-stranded and duplexed RNA in an ATP-controlled fashion. A TMV helicase polypeptide also was capable of unwinding duplexed RNA, confirming the predicted helicase function of the domain. Biochemically active helicase polypeptides were shown by gel filtration to form high-molecular-weight complexes. Electron microscopy studies revealed the presence of ring-like oligomers that displayed six-sided symmetry. Taken together, these data demonstrate that the TMV helicase domain interacts with itself to produce hexamer-like oligomers. Within the context of the full-length 126- and 183-kDa proteins, these findings suggest that the TMV replicase may form a similar oligomer.     

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.     

烟草花叶病毒（普通株中国分离物）及其弱毒苗—N14（番茄株）组织 …

用双脱氧未端经终止法对侵染性烟草共现毒普通株中国分离物（ＴＭＶ－ｖｉｒｌｇａｒ,Ｃｈｉｎｅｓｅ ｌｓｏｂｌａｔｅ,ＴＭＶ－Ｃｖ）和番茄株弱毒轩ＴＭＶ－Ｎ１４（Ａｔｔｅｎｕａｔｅｄ ＴＭＶ ｖａｃｃｉｎｅ ｓｔｒａｉｎ）基因组ｃＤＮＡｓ的核苷酸全序列进行了测定,并分析和比较了其基因组的结构和特征。结果表明：普通株基因组（Ｇｅｎｂａｎｋ接收号：ＡＦ１６５１９０）为６３９５个核苷酸：４个功能性开放阅读框     

Tobamovirus replicase coding region is involved in cell-to-cell movement

Tobacco mosaic virus (TMV) encodes a 30-kDa movement protein (MP) which enables viral movement from cell to cell. It is, however, unclear whether the 126- and 183-kDa replicase proteins are involved in the cell-to-cell movement of TMV. In the course of our studies into TMV-R, a strain with a host range different from that of TMV-U1, we have obtained an interesting chimeric virus, UR-hel. The amino acid sequence differences between UR-hel and TMV-U1 are located only in the helicase-like domain of the replicase. Interestingly, UR-hel has a defect in its cell-to-cell movement. The replication of UR-hel showed a level of replication of the genome, synthesis, and accumulation of MP similar to that observed in TMV-U1-inoculated protoplasts. Such observations support the hypothesis that the replicase coding region may in some fashion be involved in cell-to-cell movement of TMV.     

RNA helicase domain of tobamovirus replicase executes cell-to-cell movement possibly through collaboration with its nonconserved region

UR-hel, a chimeric virus obtained by replacement of the RNA helicase domain of tobacco mosaic virus (TMV)-U1 replicase with that from the TMV-R strain, could replicate similarly to TMV-U1 in protoplasts but could not move from cell to cell (K. Hirashima and Y. Watanabe, J. Virol. 75:8831-8836, 2001). It was suggested that TMV recruited both the movement protein (MP) and replicase for cell-to-cell movement by unknown mechanisms. Here, we found that a recombinant, UR-hel/V, in which the nonconserved region was derived from TMV-R in addition to the RNA helicase domain of replicase, could move from cell to cell. We also analyzed revertants isolated from UR-hel, which recovered cell-to-cell movement by their own abilities. We found amino acid substitutions responsible for phenotypic reversion only in the nonconserved region and/or RNA helicase domain but never in MP. Together, these data show that both the nonconserved region and the RNA helicase domain of replicase are involved in cell-to-cell movement. The RNA helicase domain of tobamovirus replicase possibly does not interact directly with MP but interacts with its nonconserved region to execute cell-to-cell movement.     

Replication of tobacco mosaic virus RNA

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.