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．     

紫藤种子总RNA提取方法研究

分离提取高质量的RNA是基因表达、调控和基因工程等研究的基础。植物组织中含有大量RNA酶、多糖和酚类等杂质,影响了高质量总RNA的获得。应用改良的CTAB法提取紫藤种子中的总RNA,添加了PVP、β-巯基乙醇和水饱和酚氯仿后能有效地除去大部分杂质,获得高质量的紫藤种子总RNA。3'RACE实验结果表明,以此总RNA反转录获得的cDNA为模板,可成功扩增紫藤种子中的防御素基因。     

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.     

脑表达的X连锁基因的克隆、染色体定位和初步功能研究

通过筛选人18周胎脑cDNA文库,得到一条与Bexl和Bex2在高度同源性的基因,经HUGO／GDB人类基因命名委员会的同意命名为BEX1,Northern杂交发现该基因在脑和胰腺中高表达,在心脏,胎盘,肝脏和肾脏中有较低表达,而在脑和骨骼肌中没有表达,用斯坦福大学G3辐射杂交系将BEX1定位于Xq22上的Marker DXS990和DXS 1059之间,以BEX1作为杂交探针小对小鼠的原位杂交中发现BEX1的小鼠同源基因在小鼠的生精小管中有表达,而在间质组织中没有表达,在成年小鼠（出生10周）中BEX1同源基因在生精小管的外周细胞中表达,在中层细胞（包括次级精母细胞和精子细胞）和内层细胞（主要由精子组成）中没有表达,而在6周的处于青春期的小鼠中,BEX1的同源基因在整个生精小管中都有表达,但外层细胞的表达比中层和内层细胞的表达要高得多,而在3周的幼年小鼠中,BEX1的同源基因仅有微量表达,所以BEX1在小鼠中的同源基因在青春期表达上升,生精小管成熟后表达维持在一定的水平,这提示BEX1基因可能参与精子发生及生精小管发育的过程。     

拟南芥同源转换盒基因A21反义RNA基因重组体的构建及转化

A21是拟南芥的一同源转换盒基因,以发育旺盛的组织中高度表达。采用反义RNA技术,将该基因cDNA 5′端自然410bp反向克隆到pTA7002后,构建了可在植物中调控表达A21反义RNA基因片段的重组体。以根癌农杆菌浸渍法将重组体转入拟南芥Landsberg erecta生态型植株中,筛选到转A21反义RNA基因的拟南芥植株。反义基因可在甾类激素诱导下表达出A21的反义RNA。经初步诱导表达,变异性状表现为顶端生长抑制型。     

外源RNA对小鼠成纤维细胞形态和蛋白质表达的影响

用不同RNA诱导培养小鼠成纤维细胞 ,观察外源RNA对细胞形态的影响 ,发现所添加的不同RNA均可使细胞形态发生改变。将形态改变的细胞传代 ,发现改变的细胞形态可维持至第 3代。将大鼠肝RNA诱导的小鼠成纤维细胞传代至第 4代 ,每代留样做蛋白质SDS PAGE电泳 ,观察RNA对蛋白质表达的影响 ,发现RNA诱导的细胞 ,1— 4代蛋白表达均发生了质和量的改变。     

Noncoding RNAs: Different roles in tumorigenesis

A major portion of the mammalian genome is transcribed to produce large numbers of noncoding RNAs(ncRNAs).During the past decade,the discovery of small RNAs,including the microRNAs(miRNA) and small interfering RNAs(siRNA),has led to important advances in biology.The breadth of the ncRNA field of study has substantially expanded and many recent results have revealed a range of functions that can be attributed to the miRNAs and other ncRNAs.For example,H19 RNA,HOTAIR RNA,transcribed ultraconserved regions(T-UCRs),natural antisense RNA,transfer RNA and mitochondrial noncoding RNA have been suggested to play important roles in cancers and other diseases as well as in diverse cellular processes.In this review,we focus on the current status of several classes of ncRNAs associated with cancer with the emphasis on those that are not microRNAs.