转座子沉默与DNA甲基化

转座子(transposable elements,TEs)在生物体基因组可以通过转座或逆转座移动,它拷贝数的大规模增加是基因组不稳定的重要因素,因此,维持TEs沉默是宿主进化的方向。DNA甲基化被认为是沉默TEs的可遗传表观遗传修饰方式,同时也在维持基因组稳定、基因印迹、调节基因表达中发挥作用。本研究综述了TEs对生物基因组进化和基因表达的影响,重点总结了以DNA甲基化为主的转座子沉默机制的最新研究进展,归纳了环境因素通过DNA去甲基化调控转座子跳跃的机理。图4参82     

Vernalization-mediated epigenetic silencing by a long intronic noncoding RNA

Vernalization is an environmentally-induced epigenetic switch in which winter cold triggers epigenetic silencing of floral repressors and thus provides competence to flower in spring. In Arabidopsis, winter cold triggers enrichment of tri-methylated histone H3 Lys(27) at chromatin of the floral repressor, FLOWERING LOCUS C (FLC), and results in epigenetically stable repression of FLC. This epigenetic change is mediated by an evolutionarily conserved repressive complex, polycomb repressive complex 2 (PRC2). Here, we show that a long intronic noncoding RNA [termed COLD ASSISTED INTRONIC NONCODING RNA (COLDAIR)] is required for the vernalization-mediated epigenetic repression of FLC. COLDAIR physically associates with a component of PRC2 and targets PRC2 to FLC. Our results show that COLDAIR is required for establishing stable repressive chromatin at FLC through its interaction with PRC2.     

衣藻肌动蛋白基因的克隆及核苷酸序列分析

提取衣藻总 DNA,以此为模板并参照肌动蛋白基因编码区端的序列合成寡聚核苷酸引物,进行聚合酶链式反应,扩增到一个1.2 kb 的 DNA 片段.将此片段进行克隆并经分子探针杂交,证明此片段为衣藻肌动蛋白基因.经限制性核酸内切酶处理,构建了衣藻肌动蛋白基因的物理图谱.根据物理图谱进行亚克隆以后,测得了编码区5′端的618个核苷酸的顺序.衣藻肌动蛋白基因的编码序列与高等植物之间的同源性大于90%,高于与高等动物、原生动物及真菌的同源性.但在基因的结构上,衣藻肌动蛋白基因又明显地不同于高等植物,在已经测定的基因片段上,没有发现内含子的存在.经限制性内切酶片段多态性分析,衣藻中含有一个肌动蛋白基因拷贝.     

莱茵衣藻细胞核转化系统研究进展

莱茵衣藻(Chlamydomonas reinhardti)是一种3套基因组都能进行遗传转化的真核生物.随着转基因技术的不断完善,利用衣藻作为受体生物进行外源基因表达的研究已经成为新的研究热点.特别是利用衣藻核基因组转化系统表达外源基因的研究也取得了一系列的进展.在受体衣藻品系的选择、遗传转化效率、表达调控等方面进行的系列研究,为建立一套稳定高效的外源基因表达系统打下了坚实的基础.文章对近几年在衣藻细胞核遗传转化系统等方面的研究进展进行了综述.     

Synthesis of Chlorophyllide b from Protochlorophyllide in Chlamydomonas reinhardtii y-1

In cells of Chlamydomonas reinhardtii y-1 kept in the dark, 1,7-phenanthroline stimulated the conversion of protochlorophyllide to chlorophyllide b. A membrane fraction was obtained from degreened cells that was active in this conversion only when phenanthroline was present. Untreated cells excreted protochlorophyllide, which was used as substrate for this in vitro reaction. This system may provide a clue to how chlorophyllide b is synthesized in plant cells.     

Active disruption of an RNA-protein interaction by a DExH/D RNA helicase

All aspects of cellular RNA metabolism and the replication of many viruses require DExH/D proteins that manipulate RNA in a manner that requires nucleoside triphosphates. Although DExH/D proteins have been shown to unwind purified RNA duplexes, most RNA molecules in the cellular environment are complexed with proteins. It has therefore been speculated that DExH/D proteins may also affect RNA-protein interactions. We demonstrate that the DExH protein NPH-II from vaccinia virus can displace the protein U1A from RNA in an active adenosine triphosphate-dependent fashion. NPH-II increases the rate of U1A dissociation by more than three orders of magnitude while retaining helicase processivity. This indicates that DExH/D proteins can effectively catalyze protein displacement from RNA and thereby participate in the structural reorganization of ribonucleoprotein assemblies.     

缺硫对莱茵衣藻叶绿素荧光参数和产H2速率的影响

采用TAP及TAP-S培养基培养莱茵衣藻(Chlamydomonas reinhardtii D.),测定了该藻叶绿素荧光参数及产氢气能力.结果表明:莱茵衣藻在TAP培养基内生长良好,有微量氢气产生,最高产氢速率只有1.4×10-4 ml/(mgChl·h).在TAP-S培养基内,莱茵衣藻的荧光参数Fv/Fo、ΦPSⅡ、qp分别在40～48 h下降到初始值的50%,说明缺硫对藻光合作用的影响首先发生在天线色素到PSⅡ反应中心的传能过程以及光合作用暗反应所需的酶.TAP-S培养基内藻最高产氢速率达0.22ml/(mgChl·h),缺硫可以显著提高莱茵衣藻氢气产生的速率.     

莱茵衣藻MAPK4基因RNAi载体的构建及鉴定

[目的]构建莱茵衣藻2A38的MAPK4基因的RNAi载体。[方法]提取莱茵衣藻细胞总RNA,利用PCR扩增出编码MAPK4的基因片段,用基因工程技术将MAPK4基因片段连接载体p MD18-T上,经过2次不同的双酶切后,与RNAi中间载体p T282连接,最后连接到干涉载体p Maa7IR/XIR上,用酶切及测序鉴定MAPK4基因RNAi载体并转化莱茵衣藻。[结果]经限制性内切酶酶切及测序鉴定,证实为重组质粒,并且该重组载体可在莱茵衣藻中表达。[结论]构建的MAPK4基因RNAi载体结构正确,为进一步利用RNAi技术使MAPK4基因沉默表达,研究MAPK4在莱茵衣藻中的生物学功能奠定了试验基础。     

RNA-dependent RNA polymerases,viruses, and RNA silencing

Most viruses have RNA genomes that are replicated and transcribed into messenger RNA by viral RNA-dependent RNA polymerases (RdRps), usually in concert with other viral and host factors. Many, if not most, eukaryotes also encode putative RdRps that have been implicated in sequence-specific, RNA-triggered gene silencing. Although the viral and cellular RdRps have no sequence homology, they share functional similarities such as copying messenger RNA templates and intercellular spread of the amplified sequences. Better understanding of viral and host RdRps will improve our ability to control viruses and to use RNA silencing and viruses as tools for research, biotechnology, and medicine.     

Induction and suppression of RNA silencing by an animal virus

RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Here, we show that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms.     

莱茵衣藻磷酸果糖激酶RNAi载体构建 及其对莱茵衣藻油脂积累的影响

为研究磷酸果糖激酶（PFK2）对微藻油脂积累的影响、克隆了莱茵衣藻磷酸果糖激酶同源基因CrPFK2、 构建CrPFK2 RNAi 干涉载体并转化莱茵衣藻。通过测定转基因藻在HSM 培养下的生物量和油脂含量的结果发现、 CrPFK2 RNAi 转基因藻株在HSM 培养基中生长加快、油脂含量下降了17.03%耀21.48%、说明CrPFK2 基因表达的降 低与藻细胞油脂含量减少成正相关。CrPFK2 通过改变光合碳流的多少间接调控藻细胞油脂的合成。该结果为 CrPFK2 基因应用于微藻油脂的遗传改良将起到重要作用。     

含铁蛋白FAO1融合基因的衣藻核转化表达和分析

【目的】fao1编码Candida cloacae中一种含铁辅基的长链脂肪醇氧化酶。通过构建该基因的衣藻核转化表达载体进行转化,根据表达蛋白的生物活性判断fao1能否充分利用衣藻叶绿体中的铁元素行使功能;同时也为需铁固氮酶基因的研究提供方向。【方法】通过PCR把PsaD信号肽、fao1、His-tag标签融合,连接到pDBle载体上;采用玻璃珠转化法,将重组子导入莱茵衣藻（CW15）中;经博来霉素筛选后,获得转基因植株。运用PCR和RT-PCR法检测了转化衣藻,并且用亲和层析柱纯化蛋白,同时进行FAO1活性检测。【结果】重组质粒测序完全正确;PCR和RT-PCR检测转化衣藻基因组DNA和RNA,扩增片段与预期相符;转化衣藻FAO1蛋白纯化洗脱液使ABTS（2,2′-联氨-双（3-乙基苯并噻唑啉-6-磺酸）二胺盐）底物反应变色。【结论】成功构建了信号肽、FAO1和His-tag融合基因的衣藻核转化表达载体,重组质粒已整合到莱茵衣藻基因组中,并成功转录,纯化的蛋白检测有活性。     

pH值和氮素对莱氏衣藻(Chlamydomonas reinhardtii)胞外碳酸酐酶活性的影响

对莱氏衣藻在不同ｐＨ值下胞外碳酸酐酶活性变化的观测表明,在ｐＨ７．２时诱导的酶活性最高,而ｐＨ５．５时诱导的酶活性明显低于ｐＨ７．２和ｐＨ９．０时诱导的酶活性。氮素对胞外碳酸酐酶基因表达也具有重要调控作用,在从高ＣＯ２浓度转入低ＣＯ２浓度时,缺氮或低氮浓度抑制胞外碳酸酐酶活性的增加,铵态氮诱导的酶活性高于硝态氮。     

人白细胞介素4在衣藻叶绿体中的高效转化及表达

人白细胞介素4(hIL4)是一种重要的免疫活性调节分子,与一些自身免疫性疾病有重要关系。将hIL4基因融合到衣藻叶绿体rbcL 5-’UTR和3’-UTR之间,组成5’UTR-hIL4-3’UTR片段,将其插入到来源于衣藻叶绿体基因组5.7 kb同源片段中,构建成pXhIL4同源重组质粒。利用电脉冲法将该质粒与含有aadA基因的壮观霉素抗性质粒p228共同转化衣藻叶绿体,从含100μg/mL的壮观霉素抗性平皿上筛选到阳性藻落,并通过PCR方法检测到hIL4基因,其共转化频率高达90%。经过western blotting印迹转移分析,证实了人白细胞介素4基因在衣藻叶绿体中获得成功表达,为进一步研究在衣藻叶绿体中高效表达外源蛋白奠定了基础。     

黄瓜RNA解旋酶基因家族的生物信息学鉴定和表达分析

利用生物信息学方法对黄瓜RNA解旋酶基因家族成员、基因分类、染色体定位和系统进化关系进行了分析预测,并利用实时荧光定量PCR技术检测了11个黄瓜DEAD-box RNA解旋酶基因的组织表达模式和NaCl、低温4℃、脱落酸(ABA)以及高温39℃胁迫条件下的表达变化。结果表明,黄瓜RNA解旋酶家族包含101个基因,DEAD-box、DEAH-box和DExD/H-box 3个亚家族分别包含32、27和42个基因成员;黄瓜的7条染色体均有RNA解旋酶基因分布,其中第3条染色体最多,有30个RNA解旋酶基因,仅有8个基因分布在第4条染色体上;黄瓜RNA解旋酶基因编码的蛋白包含376~2 330个氨基酸,等电点在5.13~10.00;qRT-PCR分析发现,除CsDEAD16和CsDEAD25外,其他基因在花中表达量均最高,在黄瓜根中仅检测到CsDEAD17、CsDEAD21、CsDEAD22和CsDEAD24的表达;黄瓜幼苗经上述胁迫处理后11个基因均有不同程度的表达量变化,其中CsDEAD16、CsDEAD21、CsDEAD22、CsDEAD24、CsDEAD25和CsDEAD27明显受ABA上调,而CsDEAD24的表达受高温诱导。本研究为进一步探索黄瓜RNA解旋酶基因在其生长发育中的作用和抗性作用机制,奠定了一定的理论基础。     

植物病毒编码RNA沉默抑制子的研究进展

RNA沉默是真核生物用来抵抗病毒入侵的一种普遍而又古老的防御机制，而病毒在进化过程中也相应地产生了一些与之抗衡的功能，其中编码沉默抑制子就是对抗RNA沉默的有效策略。它们分别能够在不同阶段，以不同的方式干扰来自于寄主的RNA沉默，从而有效地建立侵染。本文主要针对目前已经发现的由植物病毒编码RNA沉默抑制子的作用特征、鉴定方法以及作用副效应进行综述，并讨论了RNA沉默抑制子的研究及应用前景。     

RNA解旋酶参与植物叶片衰老调控研究进展

对植物叶片衰老机制进行了概括,并对RNA解旋酶参与叶片衰老及相关性状调控研究进展进行阐述,总结了RNA解旋酶的细胞学功能和玉米叶片衰老的研究进展,进而对RNA解旋酶基因在玉米熟期遗传改良中的应用进行了探讨。     

The DEAD-box RNA helicase Dbp5 functions in translation termination

In eukaryotes, termination of messenger RNA (mRNA) translation is mediated by the release factors eRF1 and eRF3. Using Saccharomyces cerevisiae as a model organism, we have identified a member of the DEAD-box protein (DBP) family, the DEAD-box RNA helicase and mRNA export factor Dbp5, as a player in translation termination. Dbp5 interacts genetically with both release factors and the polyadenlyate-binding protein Pab1. A physical interaction was specifically detected with eRF1. Moreover, we show that the helicase activity of Dbp5 is required for efficient stop-codon recognition, and intact Dbp5 is essential for recruitment of eRF3 into termination complexes. Therefore, Dbp5 controls the eRF3-eRF1 interaction and thus eRF3-mediated downstream events.     

Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase

Nonhexameric helicases use adenosine triphosphate (ATP) to unzip base pairs in double-stranded nucleic acids (dsNAs). Studies have suggested that these helicases unzip dsNAs in single-base pair increments, consuming one ATP molecule per base pair, but direct evidence for this mechanism is lacking. We used optical tweezers to follow the unwinding of double-stranded RNA by the hepatitis C virus NS3 helicase. Single-base pair steps by NS3 were observed, along with nascent nucleotide release that was asynchronous with base pair opening. Asynchronous release of nascent nucleotides rationalizes various observations of its dsNA unwinding and may be used to coordinate the translocation speed of NS3 along the RNA during viral replication.