登革2型病毒中国株3′非编码区RNA元件对翻译的影响

[目的]探讨登革病毒(dengue virus,DEN)3′非编码区(untranslated region,UTR)RNA元件(VR、RCS2、CS2、CS1和SL)对基因组翻译的影响.[方法]首先构建登革2′型病毒中国株(DEN2-43)UTR与萤火虫荧光素酶基因(LUC)组成的病毒诱导报告基因(virus induced reportergene,VIRG),在此基础上分别构建包含DEN2-43 3′UTR不同RNA元件的VIRG,并通过LUC检测、实时RT-PCR和Western blot方法分析含有不同元件VIRG对翻译效率的影响.[结果]发现完整的3′UTR缺失可显著抑制翻译,含有病毒VR元件VIRG的翻译水平与完整3′UTR的VIRG相似,RCS2或CS2元件可提高VIRG的翻译效率,CS1或SL元件可降低VIRG的翻译效率.[结论]DEN 2-43病毒基因组3′UTR参与了报告基因的翻译,其不同元件具有可上调和下调报告基因翻译效率的作用.     

HSV1 ICP22对HCMV主要即刻早期启动子结构的效应分析

人巨细胞病毒 (human cytomegalovirus, HCMV)主要即刻早期(major immediate-early, MIE)启动子具有很强的转录启动能力, 其顺式调控元件在这一区域形成多次重复的特殊结构, 而这些元件及其组合方式在转录启动过程中的生物学意义尚不清楚. 实验发现HSV1即刻早期蛋白ICP22能对多种病毒及细胞启动子及增强子发挥极强的转录抑制作用, 利用CAT(氯霉素乙酰转移酶)报道基因系统检测ICP22对各种不同的HCMV即刻早期基因启动子结构元件及突变体转录活性的影响显示: 尽管各种元件在ICP22存在的情况下表现出了各自独特的转录效应, 但它们组合后形成的不同突变体以及完整的HCMV启动子所表现的转录效率又不是这些元件功能的简单叠加, 并且其特定的组合方式能够拮抗ICP22的作用. 因此, 可以认为, HCMV主要即刻早期基因启动子能以特别的方式与细胞和病毒的转录因子共同调控其下游基因的表达     

一种快速准确构建酵母单杂交系统报道子的方法

酵母单杂交系统是根据DNA结合蛋白 (即转录因子 )与DNA顺式作用元件结合调控报道基因表达的原理来克隆编码目的转录因子的基因 ,其中构建带有不同DNA顺式作用元件的报道子是该系统的重要环节。目前 ,构建酵母单杂交系统报道子主要采用随机串联的方法 ,由于串联的顺式作用元件的个数和方向不能控制 ,所以筛选报道子费时费力。尝试采用同尾酶定向克隆的方法 ,将顺式作用元件按相同方向和所需的个数串联起来 ,减少了筛选报道子所需的时间和费用。     

青稞酒发酵过程中的风味功能微生物及其风味代谢特征解析

[背景]青稞酒是一个多菌种固态发酵的产物,解析发酵过程中重要的功能微生物及其代谢特征对调控青稞酒发酵具有重要作用。[目的]揭示青稞酒发酵过程中的风味功能微生物并解析其风味代谢特征。[方法]基于高通量测序技术揭示青稞酒发酵过程中的微生物群落多样性和组成;采用顶空固相微萃取结合气相色谱-质谱技术跟踪酒醅的风味信息;通过微生物属与风味物质的关联分析揭示青稞酒发酵过程中风味功能微生物菌群,并采用蒙特卡洛检验分析进一步揭示发酵过程理化因子对风味功能微生物菌群的影响;于实验室环境下重构6株微生物发酵体系,以揭示其风味代谢特征。[结果]青稞酒发酵过程中9个真菌属和8个细菌属(相对丰度>1%)占据优势,其中Aspergillus、Komagataella、Lactobacillus、Pichia、Saccharomyces和Weissella是青稞酒发酵过程主要风味功能微生物;发酵过程中还原糖(r^2=0.946 9,P=0.013 2)和酸度(r^2=0.847 6,P=0.048 6)是驱动风味功能微生物菌群演替的关键因子;6株菌的组合发酵实验揭示了体外系统与原位系统具有相同的微生物演替现象与相似的风味轮廓。[结论]研究揭示了青稞酒发酵过程关键的风味功能微生物以及驱动该菌群演替的重要环境因子,并通过组合发酵实验验证了这些微生物的风味代谢特征,为青稞酒发酵过程的风味调控提供了新的视角。     

玉米木质形成素类AGP蛋白基因家族的生物信息学分析

[目的]鉴定玉米木质形成素类AGP蛋白(Xylogen-like arabinogalactan protein,XYLP)基因家族成员,并预测分析其理化性质、蛋白结构和系统发生树。[方法]采用生物信息学方法鉴定玉米XYLP成员,对其蛋白的理化性质、疏水性、亚细胞定位、系统发生树、结构域、蛋白质二/三级结构等方面进行预测和分析。[结果]从玉米基因组中共鉴定出16个ZmXYLPs基因;其蛋白产物全长在126~304氨基酸之间,理论等电点在5.53~9.18之间,不稳定系数在25.19~55.66之间,平均疏水指数在-0.098~0.698之间,主要定位在质膜上;二级结构均由α螺旋、无规则卷曲和延伸链组成,但是三级结构各不相同。[结论]ZmXYLPs多数是定位在质膜上的疏水稳定性蛋白,其中13个ZmXYLPs和At XYLPs分别聚在两个独立进化分支中。ZmXYLPs二级结构元件的比例和相继次序各不相同,其聚合角度和空间相邻关系也存在明显差异。     

基因串联策略强化枯草芽孢杆菌终止子的功能及其在基因表达中的应用

在底盘微生物中使用高性能终止子能够显著增强基因终止效率,维持新合成mRNA的稳定性,提升外源基因表达性能。而目前缺乏专一用于枯草芽孢杆菌的终止子元件,限制了复杂功能基因电路的设计。[目的] 在枯草芽孢杆菌中挖掘新的高性能终止子,并进一步重新设计,丰富适用于这一底盘的高性能人工终止子。[方法] 将枯草芽孢杆菌终止子和枯草芽孢杆菌噬菌体终止子分别构建至终止子检测质粒中,测定各终止子的终止效率（terminator efficiency,TE）。将不同强度单终止子按强弱、强强、弱弱组合构建串联终止子,测定不同组合体的终止性能。利用高性能串联终止子进行天冬氨酸氨基裂解酶（AspA）和β-葡萄糖苷酸酶（GusA）的表达功能验证,检测高性能终止子对基因表达水平的作用。[结果] 经过检测,TB5终止子终止效率最强,TE为98%。含有TB5终止子的表达体系中GFP水平比对照上调2.2倍,RFP的表达水平下调27倍。双串联组合体终止子中,TH1.5b-TB5（TE=97%）和TB5-TB5（TE=98%）串联终止子可将RFP的表达水平下调30倍。而三串联终止子中,TB2-TB5-TB5组合体与相应的双组合体相比,GFP的表达水平虽无进一步提升,但RFP的表达仅有参照的1/300。最后,通过AspA和GusA的重组表达确证了串联终止子TH1.5b-TB5和TB10-TB5对基因表达水平的提升作用。[结论] 高终止效率的终止子可以显著提升上游基因的表达水平,通过一定规律的基因串联能够进一步提升终止子的工作效率。这种人工再设计的终止子可方便、快捷地构建至细菌基因表达系统,实现基因表达的高效调控。     

吴茱萸和臭辣吴萸果实化学成分及不同采收时间主要成分含量的变化

正吴茱萸[Evodia rutaecarpa(Juss.)Benth.]及其变种石虎[E.rutaecarpa var.officinalis(Dode)Huang]和疏毛吴茱萸[E.rutaecarpa var.bodinieri(Dode)Huang]的干燥近成熟果实均为中药材吴茱萸的原植物,可散寒止痛、降逆止呕、助阳止泻,其天然成分对心血管系统、胃肠道系统和免疫系统也有一定功效[1]171,[2-3]。吴茱萸果实主要含生物碱、苦味素和挥发油等[4],吴茱萸碱、吴茱萸次碱和柠檬苦素含量是吴茱萸药材质量的评价标准[1]172。近年来,对吴茱萸的研究多集中于其不同产地和品种的药用成分研究[5-6]。同属的臭辣吴萸(E.     

细菌磷代谢的分子调控

细菌的磷代谢通常是由一种两组分调节系统即磷酸盐调节子（Phoregulcn）所控制。所谓两组份调节系统,是指控制一大类细菌适应性反应、由结构和功能类似的两种同源蛋白族的成员组成的一类信号传导系统。其中磷酸盐调节子是研究较为深人的一种重要的两组分调节系统,它为细菌的正常磷代谢提供了保证。l细菌两组分调节系统两组分调节系统在细菌中是很常见的,目前已在十多种细菌中发现,仅在大肠杆菌中就可能有五十多种[1]。近年来,类似的传导系统在真核生物中也有发现,如酵母[2.3]和植物[4]一般说来,组成细菌两组分调节系统的两种蛋白分…     

五大连池三池沉积物中趋磁细菌多样性

[目的]探析五大连池三池沉积物中趋磁细菌(Magnetotactic bacteria,MTB)多样性及其同环境因子的关系。[方法]对沉积物的pH、总有机碳(TOC)、总氮(TN)、硫化物(S)及可溶性总铁(STI)等参数进行测定。采用磁富集和Race-track(R-T)技术收集菌体,运用基于16S rDNA V4区的高通量测序分析沉积物中MTB多样性,通过冗余分析(RDA)解读MTB多样性同环境因子的关系。[结果]沉积物pH为6.89,总氮为0.66 g·kg~(-1),总有机碳含量为7.61%,硫化物含量为1.25μg·g~(-1),可溶性总铁含量为37.5μg·g~(-1)。沉积物中MTB分属于12个门的48个属,包括趋磁螺菌、β-变形菌纲和卵球形磁细菌。RDA分析表明pH对MTB影响极显著。[结论]三池中MTB多样性较丰富,pH是影响MTB群落的重要因素。     

PCR-DGGE技术解析东北稻田蓝藻群落结构

[目的]明确东北稻田生态系统蓝藻群落结构组成变化及主要驱动因子。[方法]采用蓝藻特异性引物GC-Cya-b-F371和Cya-R783对采自东北不同土壤类型稻田土壤及田面水提取的DNA进行PCR扩增,采用DGGE电泳和条带测序技术解析稻田蓝藻群落结构。[结果]多样性指数表明,土壤中蓝藻群落结构比田面水体丰富。图谱冗余分析(RDA)显示,稻田土壤中蓝藻群落结构具有明显的地域差异,土壤碳氮比值是驱动土壤蓝藻分布的主要环境因素;而田面水体蓝藻群落结构地域差异不明显,p H值是驱动水体蓝藻分布的主要环境因素。DGGE条带测序发现,稻田蓝藻群落结构组成以丝状颤藻亚纲蓝藻(Oscillatoriophycideae)为主,一些条带基因序列与已知蓝藻相似度低于97%。[结论]东北稻田生态系统蓝藻群落结构复杂多样,生存着一些未知的蓝藻种群。     

Sequence identity of the terminal redundancies on the minus-strand DNA template is necessary but not sufficient for the template switch during hepadnavirus plus-strand DNA synthesis.

The template for hepadnavirus plus-strand DNA synthesis is a terminally redundant minus-strand DNA. An intramolecular template switch during plus-strand DNA synthesis, which permits plus-strand DNA elongation, has been proposed to be facilitated by this terminal redundancy, which is 7 to 9 nucleotides long. The aim of this study was to determine whether the presence of identical copies of the redundancy on the minus-strand DNA template was necessary and/or sufficient for the template switch and at what position(s) within the redundancy the switch occurs for duck hepatitis B virus. When dinucleotide insertions were placed within the copy of the redundancy at the 3' end of the minus-strand DNA template, novel sequences were copied into plus-strand DNA. The generation of these novel sequences could be explained by complete copying of the redundancy at the 5' end of the minus-strand DNA template followed by a template switch and then extension from a mismatched 3' terminus. In a second set of experiments, it was found that when one copy of the redundancy had either three or five nucleotides replaced the template switch was inhibited. When the identical, albeit mutant, sequences were restored in both copies of the redundancy, template switching was not necessarily restored. Our results indicate that the terminal redundancy on the minus-strand DNA template is necessary but not sufficient for template switching.     

Origin of sex

The competitive advantage of sex consists in being able to use redundancy to recover lost genetic information while minimizing the cost of redundancy. We show that the major selective forces acting early in evolution lead to RNA protocells in which each protocell contains one genome, since this maximizes the growth rate. However, damages to the RNA which block replication and failure of segregation make it advantageous to fuse periodically with another protocell to restore reproductive ability. This early, simple form of genetic recovery is similar to that occurring in extant segmented single stranded RNA viruses. As duplex DNA became the predominant form of the genetic material, the mechanism of genetic recovery evolved into the more complex process of recombinational repair, found today in a range of species. We thus conclude that sexual reproduction arose early in the evolution of life and has had a continuous evolutionary history. We cite reasons to reject arguments for gaps in the evolutionary sequence of sexual reproduction based on the presumed absence of sex in the cyanobacteria. Concerning the maintenance of the sexual cycle among current organisms, we take care to distinguish between the recombinational and outbreeding aspects of the sexual cycle. We argue that recombination, whether it be in outbreeding organisms, self-fertilizing organisms or automictic parthenogens, is maintained by the advantages of recombinational repair. We also discuss the role of DNA repair in maintaining the outbreeding aspects of the sexual cycle.     

Template switches during plus-strand DNA synthesis of duck hepatitis B virus are influenced by the base composition of the minus-strand terminal redundancy

Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3' end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. Circularization involves transfer of the nascent 3' end of the plus strand from the 5' end of the minus-strand DNA to the 3' end, where further elongation can lead to production of RC DNA. In duck hepatitis B virus (DHBV), a small terminal redundancy (5'r and 3'r) on the ends of the minus-strand DNA has been shown to be important, but not sufficient, for circularization. We investigated what contribution, if any, the base composition of the terminal redundancy made to the circularization process. Using a genetic approach, we found a strong positive correlation between the fraction of A and T residues within the terminal redundancy and the efficiency of the circularization process in those variants. Additionally, we found that the level of in situ priming increases, at the expense of primer translocation, as the fraction of A and T residues in the 3'r decreases. Thus, a terminal redundancy rich in A and T residues is important for both plus-strand template switches in DHBV.     

Optimal cDNA microarray design using expressed sequence tags for organisms with limited genomic information

Background  

Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes.     

Lack of biological significance in the 'linguistic features' of noncoding DNA--a quantitative analysis.

Recently, the application of two statistical methods (related to Zipf's distribution and Shannon's redundancy), called 'linguistic' tests, to the primary structure of DNA sequences of living organisms has excited considerable interest. Of particular importance is the claim that noncoding DNA sequences in eukaryotes display specific 'linguistic' features, being reminiscent of natural languages. Furthermore, this implies that noncoding regions of DNA may carry some new, thus far unknown, biological information which is revealed by these tests. In this paper these claims are tested quantitatively. With the aid of computer simulations of natural DNA sequences, and by applying the same 'linguistic' tests to both natural and artificial sequences, we investigate in detail the reasons of the appearance of the claimed 'linguistic' features and the associated differences between coding and noncoding DNAs. The presented results show quantitatively that the 'linguistic' tests failed to reveal any new biological information in (noncoding or coding) DNA.     

The reliability theory of aging and longevity.

Reliability theory is a general theory about systems failure. It allows researchers to predict the age-related failure kinetics for a system of given architecture (reliability structure) and given reliability of its components. Reliability theory predicts that even those systems that are entirely composed of non-aging elements (with a constant failure rate) will nevertheless deteriorate (fail more often) with age, if these systems are redundant in irreplaceable elements. Aging, therefore, is a direct consequence of systems redundancy. Reliability theory also predicts the late-life mortality deceleration with subsequent leveling-off, as well as the late-life mortality plateaus, as an inevitable consequence of redundancy exhaustion at extreme old ages. The theory explains why mortality rates increase exponentially with age (the Gompertz law) in many species, by taking into account the initial flaws (defects) in newly formed systems. It also explains why organisms "prefer" to die according to the Gompertz law, while technical devices usually fail according to the Weibull (power) law. Theoretical conditions are specified when organisms die according to the Weibull law: organisms should be relatively free of initial flaws and defects. The theory makes it possible to find a general failure law applicable to all adult and extreme old ages, where the Gompertz and the Weibull laws are just special cases of this more general failure law. The theory explains why relative differences in mortality rates of compared populations (within a given species) vanish with age, and mortality convergence is observed due to the exhaustion of initial differences in redundancy levels. Overall, reliability theory has an amazing predictive and explanatory power with a few, very general and realistic assumptions. Therefore, reliability theory seems to be a promising approach for developing a comprehensive theory of aging and longevity integrating mathematical methods with specific biological knowledge.