2株黄瓜花叶病毒卫星RNA的cDNA克隆及序列分析

在辣椒上获得2株黄瓜花叶病毒卫星RNA(CS1和CS2),通过cDNA克隆和测序并将其与35个已知的卫星RNA序列进行同源比较．结果表明,CS1和CS2的全长序列分别为336,382nt;与CS2、Rs和Yns比较,CS1从第80位起有连续30多个核苷酸的缺失．由此推测,卫星RNA的二级结构也随这些核苷酸的缺失而发生了改变．将CMV卫星RNA分为4个组,卫星RNA CS1属于中小卫星组小卫星亚组Ⅱ,CS2属于长卫星组,CS1与其他来源的卫星RNA的同源性为80．4％～93．7％,CS2与其他来源的卫星RNA的同源性则为75％～94．8％;两之间的序列同源性为86％．CMV卫星RNA的序列同源性和分组与卫星RNA分布的地区和寄主来源并无直接联系．     

小分子RNA家族中的新成员--microRNA

近来,人们在多种生物中发现了一类新的小分子RNA——microRNA(miRNA)．miRNA是20～24nt的单链RNA,在进化上具有高度的保守性,它通过与靶mRNA不完全互补配对,抑制蛋白翻译,调节内源基因表达,在基因调控中扮演了重要的角色．miRNA和siRNA(small interferenceRNA)在生成机制、作用途径等方面关系密切,它们既有区别又相互联系．小分子RNA的研究将是今后分子生物学的研究热点之一．     

基于16S rRNA序列的鳃金龟亚科5族种类的系统发育关系

测定了鳃金龟亚科部分种类的线粒体16SrRNA部分序列,运用MEGA 4.0、PAUP*4.0b10和MrBayes等软件,对5个族29个代表种的序列变异和系统关系进行了研究.序列变异分析结果显示:绢金龟族Sericini、哦鳃金龟族Hopliini、鳃金龟族Melolonthini的族内遗传距离分别为7.7%,11.3%,10.6%,族间遗传距离在12.6%～19.2%.最大似然树(ML)和贝叶斯树(BI)结果表明,所有种类分别聚集在所属的族下,哦鳃金龟族、绢金龟族分别为单系.齿爪鳃金龟属Holotrichia、云鳃金龟属Polyphylla、胸突鳃金龟属Hoplosternus、Hilyotrogus、双绺鳃金龟属Amphimallon、Hoplochelus、婆鳃金龟属Brahmina和皱鳃金龟属Trematodes聚在鳃金龟族分支下,没有形成明显的根鳃金龟族Rhizotrogini分支.齿爪鳃金龟属的非单系性与前人研究结果一致.皱鳃金龟属Trematodes和婆鳃金龟属Brahmina的代表种为姐妹种.绢金龟族的绢金龟属Serica和玛绢金龟属Maladera多数种类聚在本属分支下.证明16SrRNA序列可探讨鳃金龟亚科高级阶元的系统发育关系.     

干扰大鼠背根神经节MrgC受体mRNA的实验研究

大鼠MrgC受体与人的MrgX1受体有相似特性,但至今尚无MrgC受体的拮抗剂,阻碍了对其功能的研究.将大鼠的背根神经节(DRG)进行体外培养,加入小分子片段RNA(siRNA)对MrgC受体进行干扰.干扰时间为4 h,24 h之后再重复干扰1次.结果显示干扰后DRG中MrgC受体的mRNA表达减少84%,表明该分子片段RNA能有效使MrgC受体基因"沉默".     

Delayed resistance to Cucumber mosaic virus mediated by 3’UTR-derived hairpin RNA

RNA silencing has been shown to function in the plant antivirus defense response, leading to viral RNA degradation induced by vsiRNA-containing RISC cleavage activity. Cucumber mosaic virus （CMV） 3′UTR sequences share a high conservation of nucleotide sequence and secondary structures that are important for CMV replication. Here, in an attempt to simultaneously target the multiple genomic and subgenomic RNAs of CMV for degradation, CMV 3′UTR were used to design hairpin RNA （hpRNA） to transform tobacco （Xanthi. nc） so as to constitutively produce viral siRNAs. Most of the transgenic plants expressing CMV Q strain （Q-CMV, subgroup Ⅱ strain） RNA3 3′UTR-derived hpRNA showed delayed resistance to Q-CMV infection and exhibited recovery phenotypes. Compared with Q-CMV-inoculated leaves, the upper leaves showed weak or no disease symptoms and a reduced accumulation level of viral RNAs. Together with transient assays, our results indicate that the 3′UTR-derived siRNAs were biologically active in targeting viral RNA for degradation. Recovery resistance in transgenic plants was also observed against subgroup IB strain SD-CMV infection, indicating a broad-spectrum anti-CMV effect of the 3′UTR-based antiviral silencing. Northern blot assays indicated that there was no strong correlation between the degree of resistance and the accumulation level of 3′UTR-derived siRNAs, suggesting that to target a highly structured RNA, such as the CMV 3′UTR, the quantity of siRNAs may not be the only determinant of silencing efficiency. Target RNA secondary structures may also affect target accessibility, siRNA-containing RISC-target recognition and the consequent antiviral effect.     

兰州大尾羊脂肪组织总RNA提取方法的试验研究

在Trizol法的基础上通过优化操作规程、改进操作步骤等手段提取纯化了兰州大尾羊脂肪组织总RNA,并利用紫外分光光度计和凝胶电泳对其浓度和质量检测进行分析.结果表明:该方法可快速高效地获得适用于RT-PCR、基因表达的高通量分析、cDNA文库的构建等研究的高质量的总RNA.     

A simple and effective method for total RNA isolation of appressoria in Magnaporthe oryzae

Appressorium formation is an important event in establishing a successful interaction between the rice blast fungus, Magnaporthe oryzae, and its host plant, rice. An understanding of molecular events occurring in appressorium differentiation will give new strategies to control rice blast. A quick and reliable method to extract total RNA from appressorium is essential for studying gene expression during appressorium formation and its mechanism. We found that duplicate film is an efficient substratum for appressorium formation, even when inoculated with high density conidia. When inoculated with conidia at 1 × 106 ml^-1, the percentages of conidium germination and appressorium formation were （97.98±0.67）% and （97.88±0.45）%, respectively. We applied Trizol before appressorium collection for total RNA isolation, and as much as 113.6 lag total RNA was isolated from the mature appressoria at 24 h after inoculation. Functional analysis of two genes, MNH6 and MgATG1, isolated from the cDNA subtractive library, revealed that the quantity of RNA was good enough to construct a cDNA （complementary DNA） library or a cDNA subtractive library. This method may be also applicable for the appressorium RNA isolation of other pathogenic fungi in which conidia differentiate into appressoria in the early stages of host infection.     

高等真核生物中重叠基因研究

基因重叠是高等真核生物中一种普遍存在的基因组排现象．综述了高等真核生物中的重叠基因的分类、数量、互补或自然反义转录本（NATs）可能的生物特性和功能及重叠基因与人类疾病的关系．     

一种低危害的基因组DNA和总RNA的提取方法

从生物组织里提取基因组DNA和总RNA是多数专业研究人员都需要操作的实验内容,但该实验的常规操作流程中却存在致病性微生物的感染风险和有毒有害试剂的危害.本文从选择安全的实验材料和改进低危的实验方法两个方面入手,提出了以盐藻为实验材料的基因组DNA和总RNA的新的实验提取方法,该方法有效避免了致病性微生物的感染风险,也避免了有毒、挥发性试剂苯酚、氯仿和Trizol的使用,对核酸提取实验操作的安全性提供了一定的保障,可供研究者选取实验方案时参考.     

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2α kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2α kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2α kinases might play similar roles and that PoPKR is the predominant kinase.