植物基因组中微型反向重复转座元件研究进展

微型反向重复转座元件(miniature inverted repeat transposable element,MITE)是一类特殊的转座元件,在结构上与有缺失的DNA转座子相似,但具有反转录转座子高拷贝数的特点.MITE时常与基因相伴,对基因调控可能起重要作用,因此,MITE正逐渐成为基因和基因组进化及生物多样性研究的一种重要工具.本文综述了植物基因组MITE的结构、分类、活性及其应用研究进展.     

微生物来源的抗植物病毒活性物质研究进展

近年来,研究者利用微生物及其次生代谢产物开展了大量防治植物病毒病的研究,从中发现了许多具有抗植物病毒活性的大分子物质及小分子化合物。本文对来源于不同种类微生物的抗病毒活性物质及抗病毒机理作了论述,并对微生物来源抗植物病毒物质研究进行了展望,以期为开发用于植物病毒病防治的微生物农药提供借鉴。     

口蹄疫病毒反向遗传学研究进展

反向遗传学操作技术在口蹄疫病毒(FMDV)病原学基础研究领域的应用, 使得人们能够在基因组整体水平上研究病毒基因的功能。得益于反向遗传学系统的不断完善和发展, 目前人们对FMDV分子病原学也有了更加深入的认识和理解。本文结合实验室在FMDV反向遗传学方向上所开展的探索性研究工作, 综述了国内外利用反向遗传学操作技术在研究FMDV分子致病机制、病毒毒力与变异的关系、病毒复制的影响因素、新型FMD基因疫苗的研制等领域所取得的进展, 展望FMDV反向遗传学研究新动向。     

RNA病毒反向遗传操作技术研究进展

近年来,在分子病毒学研究领域兴起一门新型技术,即病毒的全长感染性cDNA克隆技术,是一种反向遗传操作技术(reverse genetics),通常被称为"病毒拯救(the rescue of virus)",它解决了对RNA病毒基因组难以操作这一困扰研究者多年的难题.从cDNA克隆拯救出负链RNA全病毒是20世纪90年代分子病毒学研究领域最振奋人心的突破之一,它开启了人们对病毒基因组进行人工操作和详细了解病毒基因及其产物功能的大门.该技术发展迅速,倍受国内外研究者关注[1-5].     

植物病毒卫星研究进展

植物病毒卫星研究进展周雪平,李德葆（浙江农业大学生物技术研究所,杭州３１００２９）自６０年代Ｋａｓｓａｎｉｓ发现有些烟草坏死病毒（ＴＮＶ）分离物中含有１７ｎｍ的病毒颗粒,并将其称谓卫星烟草坏死病毒（ＳＴＮＶ）以来,已发现多种植物ＲＮＡ病毒含有卫星（Ｓ...     

一个RNAi载体上反向重复片段的测序策略

相邻的反向重复DNA片段有形成单链内二级结构的倾向,属于一种测序困难的DNA模板。解决RNAi载体插入的反向重复片段的测序问题,为该类载体正确性的测序验证奠定基础。采用常规分子克隆方法构建表达小麦TaATG2串联反向重复片段的RNAi载体,设计2种策略对经菌落PCR初步鉴定的载体进行测序验证:一种是以完整的载体质粒为模板进行测序;另一种是先对载体进行酶切处理,切除反向重复片段中的一个后对保留另一个片段的线性载体进行测序。结果表明,第一种测序策略受到串联反向重复片段形成的单链内部二级结构的影响,测序信号在反向重复片段处出现衰减或乱峰,无法读取序列。第二种测序策略排除了2个反向重复片段之间的干扰,保留在载体上的片段测序信号清晰,序列准确。采用酶切切除一个片段后进行测序的方法,经过2次酶切和2次测序可以有效地对载体上的2个反向重复片段分别进行序列测定,进而确认构建载体的正确性。     

RNA病毒的反向遗传学

反向遗传操作作为一种新兴技术在RNA病毒的研究中发挥着重要作用。本文介绍了RNA病毒反向遗传学的研究方法以及RNA病毒反向遗传技术的最新研究进展。     

植物双生病毒研究进展

双生病毒（Ｇｅｍｉｎｉｖｉｒｕｓ）是一组具有双生颗粒形态的单链环状ＤＮＡ植物病毒［１～３］。典型的双生病毒为１８×３０ｎｍ颗粒,基因组大小为２．５～３．０ｋｎｔ。双生病毒研究已成为植物病毒学最活跃的领域之一。１双生病毒的性质１．１双生病毒组的成员划分...     

植物源病毒抑制剂的研究进展

天然植物提取的活性物质对植物病毒有明显的抑制作用,已经成为当今植物病毒防治的重点。对植物源病毒抑制的活性物质及作用机理的研究现状进行了综述,并将外来入侵生物用以提取病毒抑制剂的相关生物技术提出了展望。     

某些植物病毒基因组内tRNA-样结构

某些植物病毒基因组的3′末端含有能接受缬氨酸的tRNA-样结构。已经测定了它们相应区域核苷酸残基顺序,根据碱基配对原则可以折叠成不同于tRNA的二级结构,与标准tRNA^val比较时发现了某些共同的结构特点。tRNA-样结构的可能功能尚属推测。     

病毒编码基因介导的植物病毒抗性机理研究进展

根据植物自身病毒编码基因的不同,重点介绍了其病毒抗性机理的研究进展。     

植物锚蛋白研究进展

锚蛋白重复序列模体是生物体内最普遍的蛋白质序列模体之一,在多种细胞活动中主要介导蛋白质-蛋白质的相互作用。综述了近年来有关锚蛋白参与植物信号传导的研究进展。     

Functional Organization of the Inverted Repeats of IS30

The mobile element IS30 has 26-bp imperfect terminal inverted repeats (IRs) that are indispensable for transposition. We have analyzed the effects of IR mutations on both major transposition steps, the circle formation and integration of the abutted ends, characteristic for IS30. Several mutants show strikingly different phenotypes if the mutations are present at one or both ends and differentially influence the transposition steps. The two IRs are equivalent in the recombination reactions and contain several functional regions. We have determined that positions 20 to 26 are responsible for binding of the N-terminal domain of the transposase and the formation of a correct 2-bp spacer between the abutted ends. However, integration is efficient without this region, suggesting that a second binding site for the transposase may exist, possibly within the region from 4 to 11 bp. Several mutations at this part of the IRs, which are highly conserved in the IS30 family, considerably affected both major transposition steps. In addition, positions 16 and 17 seem to be responsible for distinguishing the IRs of related insertion sequences by providing specificity for the transposase to recognize its cognate ends. Finally, we show both in vivo and in vitro that position 3 has a determining role in the donor function of the ends, especially in DNA cleavage adjacent to the IRs. Taken together, the present work provides evidence for a more complex organization of the IS30 IRs than was previously suggested.Mobile DNA elements have been described in most organisms and represent a considerable proportion of their genetic material. These elements play an important role in the evolution of the host genome due to their capacities to generate DNA rearrangements and influence the expression of neighboring genes. Their ability to form compound transposons contributes to the sequestering and dispersion of accessory genes, such as those specifying resistance to antibiotics, virulence, and various catabolic activities. The simplest mobile elements are the bacterial insertion sequences (ISs), which typically harbor one or two open reading frames (ORF) coding for the transposase (Tpase). More than 2,400 ISs have been described and classified into families (IS Finder, http://www-is.biotoul.fr/) on the basis of similarities in their genetic organization and Tpases (30). The terminal inverted repeats (IRs) are essential for the transposition of most ISs. The IRs, together with the Tpase, form a complex where the cleavage and strand transfer reactions occur. The IRs generally contain two functional modules: the internal region serves as the binding site of Tpase, while the terminal part is required for DNA cleavage and the strand transfer process (2). Besides these principal cis-acting elements, some ISs carry additional regulatory DNA sequences in the IRs or in the subterminal regions (18).The IS30 family currently comprises more than 80 elements distributed throughout the Gram-positive and Gram-negative bacteria and the Archaea (IS Finder, http://www-is.biotoul.fr). IS30 (1, 5), the founding element of the family, is 1,221 bp long and has 26-bp imperfect IRs (the left end of the IR [IRL] and the right end of the IR [IRR]; Fig. ​Fig.1A)1A) and one ORF with a coding capacity for a 44.3-kDa Tpase. The element has a preference for two distinct types of target sequences: the natural hot spots (HSs), characterized by a 24-bp symmetric consensus (23), and the IRs of the element itself (21, 22). Potential helix-turn-helix motifs (HTH) responsible for HS and IR targeting are located in the N-terminal region of the Tpase (19). While the first motif, HTH1, is required only for transposition into the HS sequences, the conserved H-HTH2 motif is essential for both IR and HS targeting (15, 19).Open in a separate windowFIG. 1.Transposition assays for comparing the IS30-based transposons composed of simple IRs. (A) Comparison of the IS30 IR sequences. Dots indicate matching bases. (B) Schematic representation of the intermolecular transposition pathway. The graph shows the two major steps characteristic for IS30 transposition (steps 1 and 2). The transposon donor plasmid and its derivative, the circular transposon (thin line), carry the 26-bp IRs of IS30 (boxes with open and filled triangles representing IRL and IRR, respectively). The Cm^r gene flanking the transposon in the donor plasmid is shown as a gray box. The target plasmid (dotted line) carries the GOHS hot spot sequence (cross-hatched box). (C) Transposition frequencies of IS30-based transposons with different combinations of the IRs. The graph shows the overall frequency of transposition into the hot spot (steps 1 and 2) and the frequency of the major steps assayed separately. Data were obtained from at least three parallel experiments.IS30 transposition occurs through two major steps (14, 24) (Fig. ​(Fig.1B).1B). The first is the formation of an active intermediate by joining of the IRs. This process involves the Tpase-catalyzed cleavage of one strand at the 3′ IS end, which then attacks the same strand 2 bp outside the other IR. This strand transfer generates a single-strand bridge between the ends and leads to a figure-eight structure (33). This active transposition intermediate carrying the joined IRs probably proceeds via replicative resolution, as described for IS911 (11, 25) and IS2 (16). The resolution can lead to the circularization of a single IS or to the formation of a head-to-tail repeat of two IS30 copies. In the second step of transposition, the active forms interact with the target DNA, resulting in the known transposition products: simple insertion, deletion, inversion, or replicon fusion (14, 24).In this work, we describe the modularity of the IR ends of IS30 by analyzing several mutants. According to our results, the IS30 IRs can be divided into functional regions that are differently involved in the main transposition steps. We show that positions 2 and 3 play a pivotal role in cleavage of the ends and, consequently, in their donor function. While the terminal part (1 to 17 bp) of the IRs is indispensable for both major steps, the internal region, i.e., the binding site for the N-terminal part of Tpase (20 to 26 bp), appears to be required only for the junction formation. Although the exact role of the terminal part of IRs is less clear, several mutations in this region considerably affected both the junction formation and integration. The fact that the internal IR region is not involved in the integration suggests that the Tpase binds to other sequences during this reaction.     

DNA Inversions between Short Inverted Repeats in Escherichia Coli

Using site-specific mutagenesis in vitro, we have constructed Escherichia coli strains that allow the detection of the inversion of an 800-bp segment in the lac region. The invertible segment is bounded by inverted repeats of either 12 or 23 bp. Inversions occurring at these inverted repeats will restore the Lac+ phenotype. Inversions can be detected at both short homologies at frequencies ranging from 0.5 x 10(-8) to 1 x 10(-7). These events, which have been verified by DNA sequence analysis, are reduced up to 1000-fold in strains deficient for either RecA, RecB or RecC. They are not reduced in strains deficient in the RecF, J pathway. These results show that the RecB,C,D system can mediate rearrangements at short sequence repeats, and probably plays a major role in cellular rearrangements.     

植物转基因沉默与病毒抗性

对植物基因沉默的机制及其在植物抗病毒基因工程中的应用作了综述.植物转基因沉默是转基因植株中普遍发生的一种现象,引起植物转基因沉默的原因是多方面的,但主要涉及转录水平的基因沉默和转录后水平的基因沉默.植物对病毒的抗性机制在某些方面与基因沉默有相似之处.     

植物抗病毒病育种策略

为了得到抗病毒的寄主植物,植物育种学家进行了许多有益研究,形成了许多行之有效的抗病毒病育种策略。利用植物本身对病毒侵染所具有的一些免疫功能及其本身的一些抗性基因来获得抗性;利用来源于病毒自身基因的一些抗病性策略(PDR),如利用病毒外壳蛋白基因,病毒复制酶基因,病毒移动蛋白基因,病毒卫星RNA和反义RNA等,植物也可以获得抗性。近年来对由转录后RNA沉默引起的由RNA介导的病毒抗性策略(RMVR)也进行了深入地研究。除了PDR和RMVR以外,还有一些导致植物抗病毒的策略,包括利用美国商陆的病毒抗性蛋白(PAP),2',5’-寡腺苷酸合成酶,“植物抗体”以及病毒蛋白多肽来获得病毒抗性等。