PCR—SSCP与测序技术相结合检测小麦耐盐突变体

根据位于小麦第四同源群上与耐盐有关的gf-2.8基因的编码区序列设计1对引物,分别以两个耐盐突变体及其亲本的总DNA为模板进行PCR扩增,在5个供试材料中均扩增出1条约685bp的目的条带,SSCP电泳显示突变体974915与其他供试材料之间存在差异。测序表明冀麦24和其耐盐突变体8901-17的扩增产物序列与gf-2.8基因的发表序列相同,这表明突变体8901-17的突变位点不在该基因上,而另一耐盐突变体974915的序列中则至少存在2个单碱基突变,有一处突变导致了氨基酸的变化,该突变位点位于gf-2.8基因的保守区域内。     

翻译后修饰可能影响黄瓜花叶病毒2b 蛋白抑制子活性及稳定性

黄瓜花叶病毒 (Cucumber mosaic virus,CMV) 编码的2b蛋白具有RNA沉默抑制子功能,为了研究翻译后修饰对2b功能的影响,利用反向PCR定点突变方法对CMV-Q株系2b蛋白的1个预测的磷酸化位点 (S40) 和2个预测的泛素化/SUMO化位点 (K22,K39) 进行了点突变,同时将点突变体插入植物表达载体。通过农杆菌共注射法对3个2b突变体的抑制子活性进行了分析,结果证明,当S40突变为A (2bS40A) 后,2b抑制局部和系统沉默的活性均大幅降低;当K22突变为R (2bK2     

线粒体tRNAGlu A14693G可能是与Leber遗传性视神经病变相关的基因突变

收集了3个具有典型临床特征的中国汉族Leber遗传性视神经病变(Leber's hereditary optic neuropathy, LHON)家系。通过对先证者和家系其他成员进行眼科临床(如视力损害程度和发病年龄)检查, 发现这些家系成员中视力损害的外显率很低, 经mtDNA测序分析, 在tRNA^Glu 上发现了A14693G同质性突变位点, 多态性位点分别属于东亚单体型Y1b、Y1和Y1, 没有发现其他高度保守和有功能意义的突变位点。A14693G突变位于线粒体tRNAGlu高度保守区(通用位点为54位), 可能导致tRNA空间结构和稳定性发生改变, 继而影响tRNA的代谢, 导致线粒体蛋白合成功能受损和ATP障碍, 最终导致视力损害。所以, tRNAGlu A14693G突变可能是与视神经病变相关的致病性线粒体突变位点。     

黄瓜花叶病毒坏死型卫星RNA的cDNA克隆和全序列分析

从田间发生坏死病害的番茄病株中分离得到黄瓜花叶病毒TN分离物,经研究证实TN分离物带有1株坏死型卫星RNA(TN-SatRNA).利用已知CMV卫星RNA的两端序列作引物扩增并克隆了TN-SatRNA的cDNA.序列分析显示TN-SatRNA全长为390个核苷酸.比较TN-SatRNA与富有代表性的CMV卫星RNA的结构表明,这几种卫星RNA中具有4个结构同源区(Ⅰ:1～81nt,Ⅱ:216～261nt,Ⅲ:278～338nt;Ⅳ:349～390nt),而在82～215nt的区域几种卫星RNA的结构变化较大.在TN-SatRNA的3’端具有已报道的坏死型卫星RNA特征性序列结构.经3’RACE方法确定TN-SatRNA的3’端自然序列,与PCR扩增引物有1个核苷酸的差别.     

绒毛烟斑驳病毒卫星RNA的一种突变体的初步研究

用电泳检测VTMoV88感染绒毛烟后病毒特异性核酸组份,发现核酸的小分子RNA(卫星RNA)组成发生变化。提纯的病毒通过电镜观察及血清学鉴定并与VTMoV加以比较,证明两者在颗粒形态、大小及血清学关系上一致。以克隆的VTMoV—RNA3 cDNA片断为探针检测VTMonV与VTMoV88卫星RNA分子之间的核苷酸序列同源性,结果显示出两种毒源的卫星RNA具有高度同源性。据此,推测VTMoV88可能是卫星RNA发生变异的突变体,并对VTMoV 83卫星RNA突变体形成机制进行了初步探讨。     

特异性干扰ICP27基因的siRNA对HSV-1病毒复制的影响

针对单纯疱疹病毒1型(herpes simplex virus 1,HSV-1)的ICP27基因和其他疱疹病毒相关基因的高度保守区设计小干扰RNA(small interfering RNA,siRNA),研究其抑制病毒复制的效果。首先构建相应的小发夹RNA(small hairpin RNA,shRNA),然后通过病毒滴度测定、real-time PCR和细胞致病变效应(cytopathic effect,CPE)检测所设计的siRNA抑制病毒复制的能力。结果显示,所设计的shRNA-2(靶序列起始位置815)和shRNA-3(靶序列起始位置1 367)具有明显地抑制病毒复制的效果。尤其是shRNA-3,抑制病毒复制的效果更明显,在病毒滴度实验中,与阴性对照相比,其抑制倍数为81,同时可以下调ICP27基因的mR NA表达水平。实验结果表明shRNA-3能够显著抑制HSV-1病毒复制的能力,可以作为HSV-1感染性疾病的补充治疗手段,其对应的靶序列可以作为抗HSV-1新的靶标。     

板角山羊mtDNA Cyt b基因变异特点及系统进化研究

测定了板角山羊品种13个个体的细胞色素b基因全序列(1140 bp),比较分析了群体中细胞色素b基因的碱基组成和序列间碱基的变异情况,结果显示:在该品种(群体)中细胞色素b基因序列中6个变异位点上观察到11次T-C间和2次A-G间的碱基转换,除了有2次T-C间碱基转换发生在密码子第2位点为非同义突变以外,其余的11次碱基转换发生在密码子第3位点,均为同义突变;有1次T-G间碱基颠换发生在密码子第2位点,为非同义突变;观察到5种单倍型,单倍型多样度为0.8077.并以绵羊为外群,与山羊属其他种的同源区序列构建系统发生树.结果显示, 在系统地位上板角山羊与胃石山羊有较近的亲缘关系.     

组蛋白脱乙酰化酶—1保守氨基酸中组氨酸定点突变的建立

为了研究蛋白脱乙酰化酶－１（ＨＤＡＣ１）保守氨基酸中组氨酸的定点突变对其功能的影响,需要建立ＨＤＡＣ１保守组氨酸定点突变的突变子。在克隆野生型ＨＤＡＣ１ｃＤＮＡ的基础上,利用Ａｌ－ｔｅｒｅｄ ＳｉｔｅⅡ体外突变系统对ＨＤＡＣ１保守氨基酸中的３个组氨酸位点进行突变,并用全自动测序鉴定。结果分别获得了ＨＤＡＣ１的Ｈ１４０Ｆ、Ｈ１７８Ｆ、Ｈ１７９Ｆ的定点突变子,为进一步研究ＨＤＡＣ１保守氨基酸定点突变对     

猪繁殖与呼吸综合征病毒RNA依赖性RNA聚合酶中SDD功能区的突变分析

猪繁殖与呼吸综合征病毒PRRSV亚基因组的转录和基因组的复制由病毒复制酶引导.病毒首先合成两个多聚蛋白,随后多聚蛋白被加工分解成若干较小的非结构蛋白（nsps）,从而产生了复制酶.病毒复制酶所在的nsp9含有特异性的功能性序列模体,在正链RNA病毒的RNA依赖性RNA聚合酶RdRp中共同含有这些保守的序列模体.为了验证PRRSV所特有的SDD模体是否能够替换为其他RNA病毒相应所含有的保守模体,以及SDD的每一个氨基酸对于RdRp催化活性的影响,将其分别替换为多种不同的氨基酸.研究发现,只有将nsp9中SDD替换为GDD,即丝氨酸替换为甘氨酸S3050G时,才能拯救出病毒,并且传代后此病毒在遗传上是稳定的.改变SDD中的任何一个天门冬氨酸都对病毒是致死性的,突变后破坏了聚合酶的活性和RdRp的翻译功能,但却没有使RdRp失去复制功能.所以研究认为,SDD模体是PRRSV的RdRp所特有和保守的,不能被替换为除GDD外的其他RNA病毒所含有的保守模体,套式病毒与其他正链RNA病毒在进化上具有一定的联系.研究表明,SDD模体的两个天门冬氨酸对于PRRSV亚基因组的转录是不可缺少的;从进化上看,SDD模体可能是正链RNA病毒GDD模体的一种变异形式.     

两株黄瓜花叶病毒卫星RNA的竞争与共存研究

通过体外转录方法 ,将大小分别为 36 9nt和 385nt的 2个黄瓜花叶病毒 (Cucumbermosaicvirus,CMV)的卫星RNAYi和Yns共同与不含卫星的辅助病毒株CMV_CNa进行假重组 ,接种CMV系统寄主心叶烟。结果表明 :在接种5d的接种叶上同时检测到卫星RNA_Yi和卫星RNA_Yns;在系统叶上 ,接种 5d和 10d亦可同时检测到 2株卫星 ;但接种 15d ,在系统叶组织中只检测到卫星RNA_Yi。再将接种 5d的接种叶扩大接种到几种不同的指示植物后 ,经dsRNA抽提 ,也只获得 1条与卫星RNA_Yi大小相符的条带。通过假重组病毒株中分别获得卫星RNA并测序 ,确定2个卫星RNA的序列没有变化。卫星RNA_Yns和Yi在辅助病毒CMV_CNa作用下 ,表现出明显的竞争性 ,它们在辅助病毒中不能形成稳定的共存关系。     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。     

Support of a cucumber mosaic virus satellite RNA maps to a single amino acid proximal to the helicase domain of the helper virus.

Cucumber mosaic virus (CMV) is a tripartite RNA virus that can support the replication of satellite RNAs, small molecular parasites of the virus. Satellite RNAs can have a dramatic effect on the helper virus and the host plant in a manner specific to the helper, satellite, and host. Previously, we showed that the Sny-CMV strain is not able to support the replication of the WL1 satellite RNA in zucchini squash and that this phenotype maps to RNA 1. In the present study, we use recombinant cDNA clones of Fny- and Sny-CMV RNA 1 and a site-directed mutant of Fny-CMV RNA 1 to demonstrate that the inability to support WL1 satellite RNA maps to a single amino acid at residue 978 in the 1a protein, proximal to the helicase domain VI. Support of satellite RNA in whole plants and in protoplasts of zucchini squash is analyzed.     

Identification of a 334-ribonucleotide viral satellite as principal aetiological agent in a tomato necrosis epidemic

Cucumber mosaic virus (CMV), a widespread and economically important virus of vegetable crops, often contains a satellite RNA, here designated CARNA-5 (for CMV-associated RNA 5). Viral satellites are small nucleic acids that are sequence-unrelated to, but replicatively dependent upon, the viral genome. They essentially are molecular parasites of their helper viruses, and thereby frequently modulate viral symptom expression. Some isolates of CARNA-5 change normally moderate CMV symptoms in tomato into a lethal disease named tomato necrosis; others ameliorate CMV symptoms in tomato and other important crop plants. Here we report on the identification and molecular characterization of a 334-nucleotide necrogenic CARNA-5 isolated from tomato fields in southern Italy, where a massive outbreak of lethal necrosis occurred in the summer of 1988. This is the first time that direct evidence is given for the involvement of a viral satellite in a crop disease of epidemic scale. The possible molecular interrelationships between plant, virus, satellite and other factors that influence the satellite-induced symptom modulation underlying such a catastrophe are discussed.     

Helper virus-independent transcription and multimerization of a satellite RNA associated with cucumber mosaic virus

Satellite RNAs are the smallest infectious agents whose replication is thought to be completely dependent on their helper virus (HV). Here we report that, when expressed autonomously in the absence of HV, a variant of satellite RNA (satRNA) associated with Cucumber mosaic virus strain Q (Q-satRNA) has a propensity to localize in the nucleus and be transcribed, generating genomic and antigenomic multimeric forms. The involvement of the nuclear phase of Q-satRNA was further confirmed by confocal microscopy employing in vivo RNA-tagging and double-stranded-RNA-labeling assays. Sequence analyses revealed that the Q-satRNA multimers formed in the absence of HV, compared to when HV is present, are distinguished by the addition of a template-independent heptanucleotide motif at the monomer junctions within the multimers. Collectively, the involvement of a nuclear phase in the replication cycle of Q-satRNA not only provides a valid explanation for its persistent survival in the absence of HV but also suggests a possible evolutionary relationship to viroids that replicate in the nucleus.     

Satellite RNA-derived small interfering RNA satsiR-12 targeting the 3' untranslated region of Cucumber mosaic virus triggers viral RNAs for degradation

RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.     

Analysis of genetic bottlenecks during horizontal transmission of Cucumber mosaic virus

Genetic bottlenecks may occur in virus populations when only a few individuals are transferred horizontally from one host to another, or when a viral population moves systemically from the infection site. Genetic bottlenecks during the systemic movement of an RNA plant virus population were reported previously (H. Li and M. J. Roossinck, J. Virol. 78:10582-10587, 2004). In this study we mechanically inoculated an artificial population consisting of 12 restriction enzyme marker mutants of Cucumber mosaic virus (CMV) onto young leaves of squash plants and used two aphid species, Aphis gossypii and Myzus persicae, to transmit the virus populations from infected source plants to healthy squash plants. Horizontal transmission by aphids constituted a significant bottleneck, as the population in the aphid-inoculated plants contained far fewer mutants than the original inoculum source. Additional experiments demonstrated that genetic variation in the artificial population of CMV is not reduced during the acquisition of the virus but is significantly reduced during the inoculation period.     

Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication

Satellite RNA of Bamboo mosaic virus (satBaMV), a single-stranded mRNA type satellite encoding a protein of 20 kDa (P20), depends on the helper BaMV for replication and encapsidation. Two satBaMV isolates, BSF4 and BSL6, exhibit distinctly differential phenotypes in Nicotiana benthamiana plants when coinoculated with BaMV RNA. BSL6 significantly reduces BaMV RNA replication and suppresses the BaMV-induced symptoms, whereas BSF4 does not. By studies with chimeric satBaMVs generated by exchanging the components between BSF4 and BSL6, the genetic determinants responsible for the downregulation of BaMV replication and symptom expression were mapped at the 5' untranslated region (UTR) of BSL6. The 5' UTR of BSL6 alone is sufficient to diminish BaMV RNA replication when the 5' UTR is inserted in cis into the BaMV expression vector or when coinoculation with mutants that block the synthesis of P20 protein takes place. Further, the 5' UTR of natural satBaMV isolates contains one hypervariable (HV) region which folds into a conserved apical hairpin stem-loop (AHSL) structure (W. B. Yeh, Y. H. Hsu, H. C. Chen, and N. S. Lin, Virology 330:105-115, 2004). Interchanges of AHSL segment of HV regions between BSF4 and BSL6 led to the ability of chimeric satBaMV to interfere with BaMV replication and symptom expression. The conserved secondary structure within the HV region is a potent determinant of the downregulation of helper virus replication.     

Genetic bottlenecks reduce population variation in an experimental RNA virus population

Genetic bottlenecks are stochastic events that limit genetic variation in a population and result in founding populations that can lead to genetic drift. Evidence of past genetic bottlenecks in numerous biological systems, from mammals to viruses, has been described. In this study, we used an artificial population of Cucumber mosaic virus consisting of 12 restriction enzyme marker-bearing mutants. This population was inoculated onto young leaves of tobacco plants and monitored throughout the course of systemic infection. We show here that the genetic variation in a defined population of an RNA virus is significantly, stochastically, and reproducibly reduced during the systemic infection process, providing clear evidence of a genetic bottleneck.