Discovery of porcine microRNAs and profiling from skeletal muscle tissues during development

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沃森和克里克发表的文章的翻译

沃森和克里克发表的文章的翻译

核酸的分子结构——脱氧核糖核酸的结构我们希望证明脱氧核糖核酸盐(DNA)的结构,这种结构具有相当的生物感兴趣的新的特点。

Plauling和Corey已经提出了一种核酸结构。

他们非常友好的在其出版之前将手稿提供给我们。

他们的模型包括三个缠绕的链,与附近的糖磷酸骨架和外面的碱基。

我们认为,这种结构不理想,原因有二(1)我们相信,该材料赋予的透视图是盐,。

没有酸性氢原子,目前还不清楚是什么力量将持有的结构在一起,尤其是会互相排斥。

(2)有些距离显得有些过于小。

另三链结构也已由弗雷泽提出(在印刷中)。

在他的模型磷酸盐是在外面和内部的基础上通过氢键连接在一起。

这种结构的描述是相当不明确的,基于这个原因,我们不会对此发表评论。

我们希望提出根本不同的结构的脱氧核苷酸盐,这种结构有双螺旋且每个圈都有相同的轴线(看图)。

我们已经做出一般的化学假设,即核苷酸之间通过3'到5'磷酸二酯键连接到β- d-脱氧核糖核酸残基上。

这两条链都是右手螺旋。

但是由于这两个沿链相反的方向运行,这两条链是非常相似的。

每个链松耦合类似于Furberg的1号模型,这就是,碱基位于双螺旋的内部而磷酸盐位于外部。

糖及附近的原子结构接近Furberg 的标准配置,糖是大致垂直于与之接触的碱基。

在Z方向每一个链每隔3.4A就有一个碱基。

我们假设在同一链中相邻的核苷酸夹角为36 °,所以每条链的结构每十个核苷酸即每34A就重复一次。

一个磷原子到纵轴的间距为10A,由于磷在外面的,阳离子容易接触到他们。

其结构是一个开放的,它的水分含量是相当高的,在较低的水含量,我们希望碱基能够倾斜,这样的结构可能变得更加紧凑。

该结构新颖特点是这两条链的嘌呤和嘧啶碱基连接在一起的方式。

碱基的平面垂直于纵轴。

碱基配对,一条链的碱基和另一条链的碱基以氢键连接,因此两个碱基能够完全一致吻合,为了氢键的生成一对碱基中必须是一个是嘌呤另一个是嘧啶,碱基是如下生成的:嘌呤的1位置和嘧啶的1位置,嘌呤的6位置和嘧啶的6位置。

中国云南及西藏水生环境中的新种——棕孢香港霉(英文)

中国云南及西藏水生环境中的新种——棕孢香港霉(英文)
Hongkongmyces C.C.C. Tsang et ol., was introduced with a single species Hongkongmyces pedis, which is a human pathogen found from
菌 物 学 报 1275
BAO Dan-Feng et al. /Hongkongmyces brunneisporus sp. nov. (Lindgomycetaceae) from...
摘 要 :在大湄公河次区域的水生真菌调查中Байду номын сангаас从中国云南和西藏的沉水腐木中分离得到4 个菌株。基
于 LSU、SSU、ITS、TEFl-cx和 RPB2 序列进行多基因系统发育分析,表 明 4 个菌株属于菩提科香港霉属真
菌。系统发育分析结果显示4 个菌株聚集在一起,并与泰国香港霉形成姐妹支。基于形态学及分子系统 学研究,将 这 4 个菌株鉴定为新种棕孢香港霉。棕孢香港霉是香港霉属的第二个有性型物种,它因子囊 果的孔口处有棕色至黑色的刚毛,且子囊孢子呈梭形,孢子两端逐渐变窄且钝圆,红棕色至暗棕色,具 有多个隔膜而区别于另一个有性型物种泰国香港霉。本研究提供了该真菌新种的描述及图版并比较了该 种与其他物种的形态差异。 关 键 词 :新 种 ,形 态 学 ,系统 发 育 ,有性 型 ,分类
Supported by the National Natural Science Foundation of China (31860006, 31970021) and Fungal Diversity Conservation and Utilization Innovation Team of Dali University (ZKLX2019213). o Corresponding author. E-mail: suhongyanl6@ Received: 2020-09-23, accepted: 2020-10-29

微生物发展史

微生物发展史

微生物(microorganism,microbe)是一类体积微小、结构简单、肉眼直接看不见,必须用光学显微镜或者电子显微镜放大后才能看得见的微小生物的总称。

微生物形态结构、新陈代谢、生长繁殖及遗传变异等具有多样性,因此微生物种类繁多,在自然界中广泛分布,存在于土壤、空气、江河、湖泊,存在于动物与人的体表及其与外界相通的腔道内,如消化道、呼吸道等。

根据微生物的结构特点、遗传特性及分化组成可分为三大类。

原核细胞型微生物(prokaryote)此类微生物细胞分化低,仅有染色质组成的拟核,无核仁和核膜。

细胞质内除有核糖体外,无其它细胞器。

这类微生物按伯杰(Bergey)分类包括真细菌(eubacterium)和古细菌(archaebacterium)。

古细菌至今未发现有致病性的,因此与医学有关的原核细胞型微生物均属真细菌,包括细菌、螺旋体、衣原体、支原体、立克次体和放线菌。

真核细胞型微生物(eukaryote)这类微生物细胞核分化程度高,有核仁、核膜和染色体,胞浆内有多种细胞器,如线粒体、内质网、高尔基体等,可行有丝分裂。

包括真菌、藻类及原生动物,与医学有关的是真菌(fungus)。

非细胞型微生物这类微生物无细胞结构,仅由一种核酸和蛋白质组成。

缺乏产生能量的酶系统,必须在活细胞内增殖。

病毒(virus)属此类微生物。

自然界中绝大多数微生物对人类和动植物的生存是有益的,它们在自然界的氮、碳、硫等循环和构成生物生态环境中是必需的,对生物的繁衍及食物链的形成,微生物均起着重要作用。

微生物在人类生活和生产活动中已被广泛应用。

在农业方面,利用微生物生产细菌肥料、转基因农作物及生物杀虫剂等。

在工业方面,利用微生物发酵工程进行食品加工、酒类食醋和酱油等的酿造、抗生素生产,以及在制革、石油勘探、废物处理等生产过程中无不应用微生物。

另外,在近年发展的基因工程领域微生物也是必不可少的,例如在基因重组中,细菌的质粒、噬菌体、病毒均作为载体被广泛使用;大肠埃希菌、酵母菌等是最常用的基因工程菌。

介绍一个科学发现60字左右作文

介绍一个科学发现60字左右作文

介绍一个科学发现60字左右作文英文回答:The Discovery of CRISPR-Cas9。

The discovery of CRISPR-Cas9 is a groundbreaking scientific advancement that has revolutionized the field of genetic engineering. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9, and it is a gene-editing technology that allows scientists to make precise changes to DNA. This technology has the potential to treat a wide range of genetic diseases, as well as develop new treatments for cancer and other conditions.The discovery of CRISPR-Cas9 was made in 2012 by a team of scientists led by Jennifer Doudna and Emmanuelle Charpentier. They were studying how bacteria defend themselves against viruses, and they discovered that bacteria use CRISPR-Cas9 to cut up viral DNA. This findingled to the development of CRISPR-Cas9 as a gene-editing tool.CRISPR-Cas9 is a simple yet powerful technology. It consists of a guide RNA, which is a short piece of RNA that binds to a specific DNA sequence, and a Cas9 protein, which is an enzyme that cuts DNA. By designing the guide RNA to bind to a specific DNA sequence, scientists can use CRISPR-Cas9 to cut DNA at that specific location. This allows scientists to make precise changes to DNA, such asrepairing mutations or inserting new genes.中文回答:CRISPR-Cas9 的发现。

微生物组学英语

微生物组学英语

微生物组学英语Microbiome: The Unseen World Within UsThe human body is a complex and intricate ecosystem, teeming with trillions of microorganisms that play a vital role in our overall health and well-being. This vast and diverse community of microbes, collectively known as the microbiome, has been the subject of extensive research in recent years, as scientists strive to unravel the mysteries of this unseen world within us.The microbiome is a term that encompasses the entirety of the microbial communities that reside in various parts of the human body, including the gut, skin, oral cavity, and even the respiratory system. These microorganisms, which include bacteria, viruses, fungi, and archaea, have evolved alongside humans over millions of years, forming a symbiotic relationship that is essential for our survival.One of the most well-studied aspects of the microbiome is its role in the gut. The human gut is home to a vast and diverse array of microbes, with an estimated 100 trillion bacteria residing in thedigestive tract. These gut microbes play a crucial role in digesting and metabolizing the food we consume, extracting essential nutrients and energy that our bodies can then utilize.Beyond their role in digestion, gut microbes also have a profound impact on our immune system. They help to train and regulate the immune cells, ensuring that they are able to effectively fight off harmful pathogens while also maintaining a delicate balance that prevents autoimmune disorders. This intricate relationship between the gut microbiome and the immune system has been the focus of numerous studies, with researchers exploring the potential of probiotics and other microbial-based therapies to treat a wide range of health conditions.The skin microbiome is another area of intense research. The skin is the largest organ in the human body and is home to a diverse array of microbes, including bacteria, fungi, and viruses. These skin-dwelling microbes play a crucial role in maintaining the skin's barrier function, protecting us from harmful environmental factors and pathogens. They also contribute to the skin's overall health, helping to regulate inflammation, prevent the overgrowth of harmful microbes, and even influence the appearance of the skin.The oral microbiome is another important aspect of the human microbiome. The mouth is a complex ecosystem, with a diverse arrayof microbes that play a critical role in maintaining oral health. These microbes help to break down food, regulate pH levels, and prevent the overgrowth of harmful bacteria that can lead to dental problems such as cavities and gum disease.In addition to these well-known aspects of the microbiome, there is growing evidence that the microbial communities in other parts of the body, such as the respiratory system and the urogenital tract, also play important roles in human health and disease.One of the most exciting areas of microbiome research is the potential for microbiome-based therapies to treat a wide range of health conditions. By understanding the composition and function of the microbiome, researchers are exploring ways to manipulate it to improve human health. This includes the use of probiotics, which are live microorganisms that can be consumed to help restore the balance of the microbiome, as well as the development of personalized therapies that target specific microbial imbalances.Another promising area of research is the role of the microbiome in mental health. Emerging evidence suggests that the gut microbiome may play a significant role in the development and maintenance of mental health disorders, such as depression and anxiety. This has led to the concept of the "gut-brain axis," which posits that the bidirectional communication between the gut and the brain can havea profound impact on our emotional and cognitive well-being.As our understanding of the microbiome continues to grow, it is clear that this unseen world within us is a critical component of human health and well-being. By unraveling the complexities of the microbiome, researchers and clinicians are paving the way for new and innovative approaches to disease prevention and treatment. From improving gut health to enhancing mental well-being, the potential of the microbiome is limitless, and the future of personalized, microbiome-based medicine is rapidly taking shape.。

一键完成microRNA定量PCR引物设计

一键完成microRNA定量PCR引物设计

⼀键完成microRNA定量PCR引物设计尽管microRNA芯⽚和microRNA测序检测⽅法已经普遍使⽤,但qRT-PCR依旧是检验microRNA表达定量的⾦标准。

由于microRNA的结构特殊,长度只有18-25个碱基,⽆法直接采⽤常规的PCR技术扩增,因此RT-qPCR的引物设计对很多刚刚接触microRNA的同学都是⼀个难题。

在针对microRNA的PCR技术中,设计其引物的理念是基于延长待测microRNA的长度,构建出⼀个⾜够长的PCR模板,才能进⼀步应⽤PCR技术来定量分析。

最常⽤的microRNA 反转录PCR⽅法就是茎环法(stem-loop)和加尾法(poly-A tail)。

由于茎环法反转录的引物设计原理限制,加尾法的检测通量⽐茎环法更⾼,因此加尾法也被实验室普遍使⽤。

今天⼩编就给⼤家介绍⼀个批量设计microRNA加尾法反转录PCR引物的软件miRprimer,重⼀键完成哦!点是⼀键完成miRprimer 官⽅推荐下载⽹站:https:///projects/miRprimer/⽹站后台⽂件直接下载miRprimer地址:https:///project/miRprimer/miRprimer2_installer.zipmiRprimer2_installer.zip,整个软件的压缩包只有2.68M (2848kb)⼤⼩。

提醒:提醒1. 软件⽀持在Windows XP或更⾼系统中运⾏,还没在苹果电脑Mac OS系统测试(原因是⼩编的钱包羞涩~~)2. 经⼩编测试,最新版本miRprimer顺利运⾏,不需在电脑中安装Ruby脚本环境。

第⼀步miRprimer2_installer.zip压缩包2.68M⼤⼩,下载后解压缩⽣成同名⽂件夹,内有三个⽂件:input_miRs.txt、miRprimer2.exe、README.txt。

特别提⽰:不要更改这些⽂件的⽂件名。

特别提⽰:第⼆步input_miRs.txt,fasta格式储存的是需要设计引物的microRNA名字和序列。

分子生物学英文文献6

分子生物学英文文献6

Chapter19Detection and Quantitative Analysis of Small RNAs by PCR Seungil Ro and Wei YanAbstractIncreasing lines of evidence indicate that small non-coding RNAs including miRNAs,piRNAs,rasiRNAs, 21U endo-siRNAs,and snoRNAs are involved in many critical biological processes.Functional studies of these small RNAs require a simple,sensitive,and reliable method for detecting and quantifying levels of small RNAs.Here,we describe such a method that has been widely used for the validation of cloned small RNAs and also for quantitative analyses of small RNAs in both tissues and cells.Key words:Small RNAs,miRNAs,piRNAs,expression,PCR.1.IntroductionThe past several years have witnessed the surprising discovery ofnumerous non-coding small RNAs species encoded by genomesof virtually all species(1–6),which include microRNAs(miR-NAs)(7–10),piwi-interacting RNAs(piRNAs)(11–14),repeat-associated siRNAs(rasiRNAs)(15–18),21U endo-siRNAs(19),and small nucleolar RNAs(snoRNAs)(20).These small RNAsare involved in all aspects of cellular functions through direct orindirect interactions with genomic DNAs,RNAs,and proteins.Functional studies on these small RNAs are just beginning,andsome preliminaryfindings have suggested that they are involvedin regulating genome stability,epigenetic marking,transcription,translation,and protein functions(5,21–23).An easy and sensi-tive method to detect and quantify levels of these small RNAs inorgans or cells during developmental courses,or under different M.Sioud(ed.),RNA Therapeutics,Methods in Molecular Biology629,DOI10.1007/978-1-60761-657-3_19,©Springer Science+Business Media,LLC2010295296Ro and Yanphysiological and pathophysiological conditions,is essential forfunctional studies.Quantitative analyses of small RNAs appear tobe challenging because of their small sizes[∼20nucleotides(nt)for miRNAs,∼30nt for piRNAs,and60–200nt for snoRNAs].Northern blot analysis has been the standard method for detec-tion and quantitative analyses of RNAs.But it requires a relativelylarge amount of starting material(10–20μg of total RNA or>5μg of small RNA fraction).It is also a labor-intensive pro-cedure involving the use of polyacrylamide gel electrophoresis,electrotransfer,radioisotope-labeled probes,and autoradiogra-phy.We have developed a simple and reliable PCR-based methodfor detection and quantification of all types of small non-codingRNAs.In this method,small RNA fractions are isolated and polyAtails are added to the3 ends by polyadenylation(Fig.19.1).Small RNA cDNAs(srcDNAs)are then generated by reverseFig.19.1.Overview of small RNA complementary DNA(srcDNA)library construction forPCR or qPCR analysis.Small RNAs are polyadenylated using a polyA polymerase.ThepolyA-tailed RNAs are reverse-transcribed using a primer miRTQ containing oligo dTsflanked by an adaptor sequence.RNAs are removed by RNase H from the srcDNA.ThesrcDNA is ready for PCR or qPCR to be carried out using a small RNA-specific primer(srSP)and a universal reverse primer,RTQ-UNIr.Quantitative Analysis of Small RNAs297transcription using a primer consisting of adaptor sequences atthe5 end and polyT at the3 end(miRTQ).Using the srcD-NAs,non-quantitative or quantitative PCR can then be per-formed using a small RNA-specific primer and the RTQ-UNIrprimer.This method has been utilized by investigators in numer-ous studies(18,24–38).Two recent technologies,454sequenc-ing and microarray(39,40)for high-throughput analyses of miR-NAs and other small RNAs,also need an independent method forvalidation.454sequencing,the next-generation sequencing tech-nology,allows virtually exhaustive sequencing of all small RNAspecies within a small RNA library.However,each of the clonednovel small RNAs needs to be validated by examining its expres-sion in organs or in cells.Microarray assays of miRNAs have beenavailable but only known or bioinformatically predicted miR-NAs are covered.Similar to mRNA microarray analyses,the up-or down-regulation of miRNA levels under different conditionsneeds to be further validated using conventional Northern blotanalyses or PCR-based methods like the one that we are describ-ing here.2.Materials2.1.Isolation of Small RNAs, Polyadenylation,and Purification 1.mirVana miRNA Isolation Kit(Ambion).2.Phosphate-buffered saline(PBS)buffer.3.Poly(A)polymerase.4.mirVana Probe and Marker Kit(Ambion).2.2.Reverse Transcription,PCR, and Quantitative PCR 1.Superscript III First-Strand Synthesis System for RT-PCR(Invitrogen).2.miRTQ primers(Table19.1).3.AmpliTaq Gold PCR Master Mix for PCR.4.SYBR Green PCR Master Mix for qPCR.5.A miRNA-specific primer(e.g.,let-7a)and RTQ-UNIr(Table19.1).6.Agarose and100bp DNA ladder.3.Methods3.1.Isolation of Small RNAs 1.Harvest tissue(≤250mg)or cells in a1.7-mL tube with500μL of cold PBS.T a b l e 19.1O l i g o n u c l e o t i d e s u s e dN a m eS e q u e n c e (5 –3 )N o t eU s a g em i R T QC G A A T T C T A G A G C T C G A G G C A G G C G A C A T G G C T G G C T A G T T A A G C T T G G T A C C G A G C T A G T C C T T T T T T T T T T T T T T T T T T T T T T T T T V N ∗R N a s e f r e e ,H P L CR e v e r s e t r a n s c r i p t i o nR T Q -U N I r C G A A T T C T A G A G C T C G A G G C A G GR e g u l a r d e s a l t i n gP C R /q P C Rl e t -7a T G A G G T A G T A G G T T G T A T A G R e g u l a r d e s a l t i n gP C R /q P C R∗V =A ,C ,o r G ;N =A ,C ,G ,o r TQuantitative Analysis of Small RNAs299 2.Centrifuge at∼5,000rpm for2min at room temperature(RT).3.Remove PBS as much as possible.For cells,remove PBScarefully without breaking the pellet,leave∼100μL of PBS,and resuspend cells by tapping gently.4.Add300–600μL of lysis/binding buffer(10volumes pertissue mass)on ice.When you start with frozen tissue or cells,immediately add lysis/binding buffer(10volumes per tissue mass)on ice.5.Cut tissue into small pieces using scissors and grind it usinga homogenizer.For cells,skip this step.6.Vortex for40s to mix.7.Add one-tenth volume of miRNA homogenate additive onice and mix well by vortexing.8.Leave the mixture on ice for10min.For tissue,mix it every2min.9.Add an equal volume(330–660μL)of acid-phenol:chloroform.Be sure to withdraw from the bottom phase(the upper phase is an aqueous buffer).10.Mix thoroughly by inverting the tubes several times.11.Centrifuge at10,000rpm for5min at RT.12.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.13.Measure the volume using a scale(1g=∼1mL)andnote it.14.Add one-third volume of100%ethanol at RT to the recov-ered aqueous phase.15.Mix thoroughly by inverting the tubes several times.16.Transfer up to700μL of the mixture into afilter cartridgewithin a collection bel thefilter as total RNA.When you have>700μL of the mixture,apply it in suc-cessive application to the samefilter.17.Centrifuge at10,000rpm for15s at RT.18.Collect thefiltrate(theflow-through).Save the cartridgefor total RNA isolation(go to Step24).19.Add two-third volume of100%ethanol at RT to theflow-through.20.Mix thoroughly by inverting the tubes several times.21.Transfer up to700μL of the mixture into a newfilterbel thefilter as small RNA.When you have >700μL of thefiltrate mixture,apply it in successive appli-cation to the samefilter.300Ro and Yan22.Centrifuge at10,000rpm for15s at RT.23.Discard theflow-through and repeat until all of thefiltratemixture is passed through thefilter.Reuse the collectiontube for the following washing steps.24.Apply700μL of miRNA wash solution1(working solu-tion mixed with ethanol)to thefilter.25.Centrifuge at10,000rpm for15s at RT.26.Discard theflow-through.27.Apply500μL of miRNA wash solution2/3(working solu-tion mixed with ethanol)to thefilter.28.Centrifuge at10,000rpm for15s at RT.29.Discard theflow-through and repeat Step27.30.Centrifuge at12,000rpm for1min at RT.31.Transfer thefilter cartridge to a new collection tube.32.Apply100μL of pre-heated(95◦C)elution solution orRNase-free water to the center of thefilter and close thecap.Aliquot a desired amount of elution solution intoa1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap carefully because it might splashdue to pressure buildup.33.Leave thefilter tube alone for1min at RT.34.Centrifuge at12,000rpm for1min at RT.35.Measure total RNA and small RNA concentrations usingNanoDrop or another spectrophotometer.36.Store it at–80◦C until used.3.2.Polyadenylation1.Set up a reaction mixture with a total volume of50μL in a0.5-mL tube containing0.1–2μg of small RNAs,10μL of5×E-PAP buffer,5μL of25mM MnCl2,5μL of10mMATP,1μL(2U)of Escherichia coli poly(A)polymerase I,and RNase-free water(up to50μL).When you have a lowconcentration of small RNAs,increase the total volume;5×E-PAP buffer,25mM MnCl2,and10mM ATP should beincreased accordingly.2.Mix well and spin the tube briefly.3.Incubate for1h at37◦C.3.3.Purification 1.Add an equal volume(50μL)of acid-phenol:chloroformto the polyadenylation reaction mixture.When you have>50μL of the mixture,increase acid-phenol:chloroformaccordingly.2.Mix thoroughly by tapping the tube.Quantitative Analysis of Small RNAs3013.Centrifuge at10,000rpm for5min at RT.4.Recover the aqueous phase carefully without disrupting thelower phase and transfer it to a fresh tube.5.Add12volumes(600μL)of binding/washing buffer tothe aqueous phase.When you have>50μL of the aqueous phase,increase binding/washing buffer accordingly.6.Transfer up to460μL of the mixture into a purificationcartridge within a collection tube.7.Centrifuge at10,000rpm for15s at RT.8.Discard thefiltrate(theflow-through)and repeat until allof the mixture is passed through the cartridge.Reuse the collection tube.9.Apply300μL of binding/washing buffer to the cartridge.10.Centrifuge at12,000rpm for1min at RT.11.Transfer the cartridge to a new collection tube.12.Apply25μL of pre-heated(95◦C)elution solution to thecenter of thefilter and close the cap.Aliquot a desired amount of elution solution into a1.7-mL tube and heat it on a heat block at95◦C for∼15min.Open the cap care-fully because it might be splash due to pressure buildup.13.Let thefilter tube stand for1min at RT.14.Centrifuge at12,000rpm for1min at RT.15.Repeat Steps12–14with a second aliquot of25μL ofpre-heated(95◦C)elution solution.16.Measure polyadenylated(tailed)RNA concentration usingNanoDrop or another spectrophotometer.17.Store it at–80◦C until used.After polyadenylation,RNAconcentration should increase up to5–10times of the start-ing concentration.3.4.Reverse Transcription 1.Mix2μg of tailed RNAs,1μL(1μg)of miRTQ,andRNase-free water(up to21μL)in a PCR tube.2.Incubate for10min at65◦C and for5min at4◦C.3.Add1μL of10mM dNTP mix,1μL of RNaseOUT,4μLof10×RT buffer,4μL of0.1M DTT,8μL of25mM MgCl2,and1μL of SuperScript III reverse transcriptase to the mixture.When you have a low concentration of lig-ated RNAs,increase the total volume;10×RT buffer,0.1M DTT,and25mM MgCl2should be increased accordingly.4.Mix well and spin the tube briefly.5.Incubate for60min at50◦C and for5min at85◦C toinactivate the reaction.302Ro and Yan6.Add1μL of RNase H to the mixture.7.Incubate for20min at37◦C.8.Add60μL of nuclease-free water.3.5.PCR and qPCR 1.Set up a reaction mixture with a total volume of25μL ina PCR tube containing1μL of small RNA cDNAs(srcD-NAs),1μL(5pmol of a miRNA-specific primer(srSP),1μL(5pmol)of RTQ-UNIr,12.5μL of AmpliTaq GoldPCR Master Mix,and9.5μL of nuclease-free water.ForqPCR,use SYBR Green PCR Master Mix instead of Ampli-Taq Gold PCR Master Mix.2.Mix well and spin the tube briefly.3.Start PCR or qPCR with the conditions:95◦C for10minand then40cycles at95◦C for15s,at48◦C for30s and at60◦C for1min.4.Adjust annealing Tm according to the Tm of your primer5.Run2μL of the PCR or qPCR products along with a100bpDNA ladder on a2%agarose gel.∼PCR products should be∼120–200bp depending on the small RNA species(e.g.,∼120–130bp for miRNAs and piRNAs).4.Notes1.This PCR method can be used for quantitative PCR(qPCR)or semi-quantitative PCR(semi-qPCR)on small RNAs suchas miRNAs,piRNAs,snoRNAs,small interfering RNAs(siRNAs),transfer RNAs(tRNAs),and ribosomal RNAs(rRNAs)(18,24–38).2.Design miRNA-specific primers to contain only the“coresequence”since our cloning method uses two degeneratenucleotides(VN)at the3 end to make small RNA cDNAs(srcDNAs)(see let-7a,Table19.1).3.For qPCR analysis,two miRNAs and a piRNA were quan-titated using the SYBR Green PCR Master Mix(41).Cyclethreshold(Ct)is the cycle number at which thefluorescencesignal reaches the threshold level above the background.ACt value for each miRNA tested was automatically calculatedby setting the threshold level to be0.1–0.3with auto base-line.All Ct values depend on the abundance of target miR-NAs.For example,average Ct values for let-7isoforms rangefrom17to20when25ng of each srcDNA sample from themultiple tissues was used(see(41).Quantitative Analysis of Small RNAs3034.This method amplifies over a broad dynamic range up to10orders of magnitude and has excellent sensitivity capable ofdetecting as little as0.001ng of the srcDNA in qPCR assays.5.For qPCR,each small RNA-specific primer should be testedalong with a known control primer(e.g.,let-7a)for PCRefficiency.Good efficiencies range from90%to110%calcu-lated from slopes between–3.1and–3.6.6.On an agarose gel,mature miRNAs and precursor miRNAs(pre-miRNAs)can be differentiated by their size.PCR prod-ucts containing miRNAs will be∼120bp long in size whileproducts containing pre-miRNAs will be∼170bp long.However,our PCR method preferentially amplifies maturemiRNAs(see Results and Discussion in(41)).We testedour PCR method to quantify over100miRNAs,but neverdetected pre-miRNAs(18,29–31,38). 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英文文献阅读笔记

英文文献阅读笔记

英文文献阅读笔记Title: The Role of MicroRNAs in Cancer Development and ProgressionAuthor: Kaitlyn SmithPublication: Cancer Research JournalDate: January 2023Summary:This article delves into the intricate world of microRNAs (miRNAs) and their critical role in cancer development and progression. miRNAs are small non-coding RNA molecules that regulate gene expression, playing a significant role in various biological processes including cell growth, differentiation, and apoptosis. The article highlights the complex interplay between miRNAs and cancer, discussing how these tiny regulators can act as either tumor suppressors or oncogenes, depending on the context.Key Points:1. miRNAs function as post-transcriptional regulators, binding to the 3' untranslated region (3'UTR) of target mRNAs, leading to either mRNA degradation or translational repression.2. Dysregulation of miRNAs is common in cancer, often characterized by miRNA overexpression or underexpression. These changes can lead to abnormal gene expression patterns that drive cancer development and progression.3. miRNAs can act as tumor suppressors by targeting oncogenes for degradation or inhibiting their translation, or they can function as oncogenes by targeting tumor suppressor genes.4. Therapeutic potential of miRNAs has been recognized, with several ongoing clinical trials exploring the use of miRNA-based drugs or inhibitors for the treatment of various cancers.5. The role of miRNAs in cancer is further complicated by their ability to influence the tumor microenvironment, including immune cell infiltration and stromal cell activation.6. Future research directions include understanding the precise mechanisms of miRNA regulation in cancer, identifying specific miRNA signatures predictive of tumor behavior and patientprognosis, and developing more effective miRNA-based therapeutic strategies.Reflections:This article has significantly expanded my understanding of the complex role of miRNAs in cancer. The concept of miRNAs functioning as both tumor suppressors and oncogenes is fascinating and underscores the remarkable versatility of these tiny regulators. The therapeutic potential of miRNAs is also promising, offering new avenues for cancer treatment. However, the challenges associated with developing effective miRNA-based therapies are numerous, requiring a more comprehensive understanding of miRNA biology and the tumor microenvironment.。

miRNAs对肠道健康的调控作用及机理

miRNAs对肠道健康的调控作用及机理

miRNAs对肠道健康的调控作用及机理陶新;徐子伟【摘要】本文在介绍肠道组织miRNAs表达的基础上,主要就miRNAs的差异表达对肠道上皮细胞增殖、分化、凋亡的影响以及miRNAs在肠道黏膜免疫、抵御外界病原感染、抗应激、调节肠道营养代谢和维持肠道稳态等方面所起的作用及其机理进行了综述.【期刊名称】《动物营养学报》【年(卷),期】2013(025)009【总页数】5页(P1911-1915)【关键词】miRNAs;肠道;肠道上皮细胞;肠道黏膜免疫;肠道稳态【作者】陶新;徐子伟【作者单位】浙江省农业科学院畜牧兽医研究所,杭州310021;浙江省农业科学院畜牧兽医研究所,杭州310021【正文语种】中文【中图分类】S852.2miRNAs是一类长约18~26 nt的内源性单链非编码小分子RNA,作为细胞增殖、分化和凋亡的关键调控因子,影响着机体内部几乎所有的信号通路。

它是体内最大的一类基因表达调控因子,可调控机体内超过1/3蛋白编码基因的表达,在动物的生长、发育和疾病发生发展过程中起着重要作用。

miRNAs参与了干细胞的自我更新和多向分化[1],与免疫、消化、神经、内分泌和心血管等系统的发育和疾病的发生发展有着密切关系。

miRNAs的表达模式具有时序性和组织特异性,但大多数的miRNAs在相关物种间又高度保守,提示它们在关键细胞进程如应激适应能力和激素信号传导中起着重要作用[2]。

目前有关miRNAs在肠道组织中的报道主要集中在其差异性表达与结直肠癌[3-5]、炎症性肠病(IBD)[6]、肠易激综合征(IBS)[7]和囊性纤维化(CF)[8]等肠道疾病的发生、发展及转归关系的研究。

然而越来越多的研究结果表明,miRNAs的表达同样对肠道形态和结构的发育进程、正常维持以及肠道各种功能的发挥等起着重要调控作用。

因此,全面而深入地了解miRNAs在肠道组织中的表达和调控机理,对于深层次挖掘和揭示影响机体肠道健康的分子机理具有非常重要的科学意义。

MicroRNA在非洲猪瘟研究中的应用

MicroRNA在非洲猪瘟研究中的应用

动物医学进展,02 ,42(4)=120-123Progress in Veterinary MedicineMicroRNA 在非洲猪瘟研究中的应用张兆博1,张思诗2,汉可欣1,李晓易3,文雪霞1,汉丽梅1宀(.沈阳农业大学畜牧兽医学院,辽宁沈阳1 10866; 2.中国农业科学院上海兽医研究所,上海20024 1 ; 3.长春师范大学,吉林长春130031; 4.东北畜禽疫病研究教育部重点实验室,辽宁沈阳1 10866)摘 要:microRNA 是非编码RNA 中一种重要的不具有编码能力的调节RNA ,它广泛存在于真核生物体内并对机体的发育、细胞凋亡、基因表达及蛋白质表达等一系列生物学过程起调控作用。

非洲猪瘟(Af-rican swine fever , ASF)是一种传染性强、危害大的急性传染病,已严重危害我国养猪业的正常发展。

对 ASF 发病过程中microRNA 的研究已成为近年来ASF 的研究热点之一。

论文对近年来ASF 发病过程中的microRNA 的作用及其机制进行综述,为未来microRNA 在ASF 防控中发挥作用提供参考。

关键词:非洲猪瘟;microRNA ;非洲猪瘟病毒中图分类号:S852.651非洲猪瘟(African swine fever, ASF )是由非洲 猪瘟病毒(African swine fever virus , ASFV )感染引起的急性、热性传染病,家猪和野猪都为该病的易感 对象。

ASFV 是一种20面体、有囊膜的双链DNA病毒,不同来源的分离株病毒基因组长度在170 kb~193 kb 之间[1]。

ASFV 约有151个〜167个开放阅读框(open reading frame, ORF )编码5个多基因家族,但目前部分ORF 编码蛋白的功能尚未明 确⑵。

ASFV 的传播方式多样,其既可以通过易感 动物之间的直接接触传播,也可以通过污染的猪肉、毒蛇、车辆、人及扁虱等途径间接传播[]。

细叶石斛的位点特异性PCR鉴别(英文)

细叶石斛的位点特异性PCR鉴别(英文)

细叶石斛的位点特异性PCR鉴别(英文)
丁小余;张卫明;保曙琳;常俊
【期刊名称】《中国医学生物技术应用》
【年(卷),期】2002(000)004
【摘要】根据细叶石斛及其它37种枫斗类和黄草类石斛的rDNAITS序列,我们设计了位点特异性PCR鉴别引物XY-JB01S和XY-JB01X,对细叶石斛进行了成功的DNA分子鉴别。

在进行位点特异性鉴别PCR之前,首先运用扩增ITS区的通用引物P1、P2对模板DNA进行扩增,以验证模板的可靠性和扩增的合适浓度。

当退火温度上升为64℃,只有细叶石斛的模板DNA能被扩增出来,而其它的37种石斛属植物均为阴性。

该鉴别反应重复性好,已在鉴别细叶石斛中发挥重要作用。

与DNA 测序鉴别方法相比,位点特异性PCR具有简单、省时、高效、准确等优点。

【总页数】8页(P36-43)
【作者】丁小余;张卫明;保曙琳;常俊
【作者单位】南京师范大学生命科学学院资源生物学重点实验室;南京野生植物综合利用研究院;南京师范大学生命科学学院资源生物学重点实验室;南京210097;南京210042;南京210097;南京210097
【正文语种】中文
【中图分类】R284
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益生菌肠道微生物的基因组学英文论文及翻译

益生菌肠道微生物的基因组学英文论文及翻译

The genomics of probiotic intestinal microorganismsSeppo Salminen1 , Jussi Nurmi2 and Miguel Gueimonde1(1) Functional Foods Forum, University of Turku, FIN-20014 Turku, Finland(2) Department of Biotechnology, University of Turku, FIN-20014 Turku, FinlandSeppo SalminenEmail: *********************Published online: 29 June 2005AbstractAn intestinal population of beneficial commensal microorganisms helps maintain human health, and some of these bacteria have been found to significantly reduce the risk of gut-associated disease and to alleviate disease symptoms. The genomic characterization of probiotic bacteria and other commensal intestinal bacteria that is now under way will help to deepen our understanding of their beneficial effects.While the sequencing of the human genome [1, 2] has increased ourunderstanding of the role of genetic factors in health and disease, each human being harbors many more genes than those in their own genome. These belong to our commensal and symbiotic intestinal microorganisms - our intestinal 'microbiome' - which play an important role in maintaining human health and well-being. A more appropriate image of ourselves would be drawn if the genomes of our intestinal microbiota were taken into account. The microbiome may contain more than 100 times the number of genes in the human genome [3] and provides many functions that humans have thus not needed to develop themselves. The indigenous intestinal microbiota provides a barrier against pathogenic bacteria and other harmful food components [4–6]. It has also been shown to have a direct impact on the morphology of the gut [7], and many intestinal diseases can be linked to disturbances in the intestinal microbial population [8].The indigenous microbiota of an infant's gastrointestinal tract is originally created through contact with the diverse microbiota of the parents and the immediate environment. During breast feeding, initial microbial colonization is enhanced by galacto-oligosaccharides in breast milk and contact with the skin microbiota of the mother. This early colonization process directs the microbial succession until weaning and forms the basis for a healthy microbiota. The viable microbes in the adultintestine outnumber the cells in the human body tenfold, and the composition of this microbial population throughout life is unique to each human being. During adulthood and aging the composition and diversity of the microbiota can vary as a result of disease and the genetic background of the individual.Current research into the intestinal microbiome is focused on obtaining genomic data from important intestinal commensals and from probiotics, microorganisms that appear to actively promote health. This genomic information indicates that gut commensals not only derive food and other growth factors from the intestinal contents but also influence their human hosts by providing maturational signals for the developing infant and child, as well as providing signals that can lead to an alteration in the barrier mechanisms of the gut. It has been reported that colonization by particular bacteria has a major role in rapidly providing humans with energy from their food [9]. For example, the intestinal commensal Bacteroides thetaiotaomicron has been shown to have a major role in this process, and whole-genome transcriptional profiling of the bacterium has shown that specific diets can be associated with selective upregulation of bacterial genes that facilitate delivery of products of carbohydrate breakdown to the host's energy metabolism [10, 11]. Key microbial groups in the intestinal microbiota are highly flexible in adapting to changes in diet, and thus detailed prediction of their actions and effects may be difficult. Although genomic studies have revealed important details about the impact of the intestinal microbiota on specific processes [3, 11–14], the effects of species composition and microbial diversity and their potential compensatory functions are still not understood.Probiotics and healthA probiotic has been defined by a working group of the International Life Sciences Institute Europe (ILSI Europe) as "a viable microbial food supplement which beneficially influences the health of the host" [15]. Probiotics are usually members of the healthy gut microbiota and their addition can assist in returning a disturbed microbiota to its normal beneficial composition. The ILSI definition implies that safety and efficacy must be scientifically demonstrated for each new probiotic strain and product. Criteria for selecting probiotics that are specific for a desired target have been developed, but general criteria that must be satisfied include the ability to adhere to intestinal mucosa and tolerance of acid and bile. Such criteria have proved useful but cumbersome in current selection processes, as there are several adherence mechanisms and they influence gene upregulation differently in the host. Therefore, two different adhesion studies need to be conducted on each strain and theirpredictive value for specific functions is not always good or optimal. Demonstration of the effects of probiotics on health includes research on mechanisms and clinical intervention studies with human subjects belonging to target groups.The revelation of the human genome sequence has increased our understanding of the genetic deviations that lead to or predispose to gastrointestinal disease as well as to diseases associated with the gut, such as food allergies. In 1995, the first genome of a free-living organism, the bacterium Haemophilus influenzae, was sequenced [16]. Since then, over 200 bacterial genome sequences, mainly of pathogenic microorganisms, have been completed. The first genome of a mammalian lactic-acid bacterium, that of Lactococcus lactis, a microorganism of great industrial interest, was completed in 2001 [17]. More recently, the genomes of numerous other lactic-acid bacteria [18], bifidobacteria [12] and other intestinal microorganisms [13, 19, 20] have been sequenced, and others are under way [21]. Table 1lists the probiotic bacteria that have been sequenced. These great breakthroughs have demonstrated that evolution has adapted both microbes and humans to their current state of cohabitation, or even symbiosis, which is beneficial to both parties and facilitates a healthy and relatively stable but adaptable gut environment.Table 1Lessons from genomesLactic-acid bacteria and bifidobacteria can act as biomarkers of gut health by giving early warning of aberrations that represent a risk of specific gut diseases. Only a few members of the genera Lactobacillus and Bifidobacterium, two genera that provide many probiotics, have been completely sequenced. The key issue for the microbiota, for probiotics, and for their human hosts is the flexibility of the microorganisms in coping with a changeable local environment and microenvironments.This flexibility is emphasized in the completed genomes of intestinal and probiotic microorganisms. The complete genome sequence of the probiotic Lactobacillus acidophilus NCFM has recently been published by Altermann et al. [22]. The genome is relatively small and the bacterium appears to be unable to synthesize several amino acids, vitamins and cofactors. Italso encodes a number of permeases, glycolases and peptidases for rapid uptake and utilization of sugars and amino acids from the human intestine, especially the upper gastrointestinal tract. The authors also report a number of cell-surface proteins, such as mucus- and fibronectin-binding proteins, that enable this strain to adhere to the intestinal epithelium and to exchange signals with the intestinal immune system. Flexibility is guaranteed by a number of regulatory systems, including several transcriptional regulators, six PurR-type repressors and ninetwo-component systems, and by a variety of sugar transporters. The genome of another probiotic, Lactobacillus johnsonii [23], also lacks some genes involved in the synthesis of amino acids, purine nucleotides and numerous cofactors, but contains numerous peptidases, amino-acid permeases and other transporters, indicating a strong dependence on the host.The presence of bile-salt hydrolases and transporters in these bacteria indicates an adaptation to the upper gastrointestinal tract [23], enabling the bacteria to survive the acidic and bile-rich environments of the stomach and small intestine. In this regard, bile-salt hydrolases have been found in most of the sequenced genomes of bifidobacteria and lactic-acid bacteria [24], and these enzymes can have a significant impact on bacterial survival. Another lactic-acid bacterium, Lactobacillus plantarum WCFS1, also contains a large number of genes related to carbohydrate transport and utilization, and has genes for the production of exopolysaccharides and antimicrobial agents [18], indicating a good adaptation to a variety of environments, including the human small intestine [14]. In general, flexibility and adaptability are reflected by a large number of regulatory and transport functions.Microorganisms that inhabit the human colon, such as B. thetaiotaomicron and Bifidobacterium longum [12], have a great number of genes devoted to oligosaccharide transport and metabolism, indicating adaptation to life in the large intestine and differentiating them from, for example, L. johnsonii [23]. Genomic research has also provided initial information on the relationship between components of the diet and intestinal microorganisms. The genome of B. longum [12] suggests the ability to scan for nutrient availability in the lower gastrointestinal tract in human infants. This strain is adapted to utilizing the oligosaccharides in human milk along with intestinal mucins that are available in the colon of breast-fed infants. On the other hand, the genome of L. acidophilus has a gene cluster related to the metabolism of fructo-oligosaccharides, carbohydrates that are commonly used as prebiotics, or substrates to肠道微生物益生菌的基因组学塞波萨米宁,尤西鲁米和米格尔哥尔摩得(1)功能性食品论坛,图尔库大学,FIN-20014芬兰图尔库(2)土尔库大学生物技术系,FIN-20014芬兰图尔库塞波萨米宁电子邮件:seppo.salminen utu.fi线上发表于2005年6月29日摘要肠道有益的共生微生物有助于维护人体健康,一些这些细菌被发现显着降低肠道疾病的风险和减轻疾病的症状。

蒲英语作文

蒲英语作文

探索蒲公英的奥秘:一种微小却强大的自然之力In the vast tapestry of nature, the humble dandelion often goes unnoticed, overshadowed by its more ostentatious neighbors. However, this unassuming plant holds within it the secrets of resilience, adaptability, and survival, qualities that are not only fascinating but also serve as powerful metaphors for our own lives.The dandelion, with its bright yellow flowers and lush green leaves, is a familiar sight in gardens, fields, and even on the sides of roads. Its ability to thrive in a wide range of environments, from fertile soil to barren patches, is remarkable. It is a symbol of tenacity, growing where others might not, persisting in the face of adversity.What many people fail to see is the remarkable journey that the dandelion embarks on after its flowers fade. The seed heads, which contain hundreds of tiny seeds, are dispersed by the wind, each seed carrying the potential for new life. This dispersal mechanism is not only a brilliant survival strategy but also a beautiful display of nature's elegance and precision.The seeds, light and airy, are carried by the slightest breeze, landing in unexpected places where they take root and begin the cycle of life anew. This amazing journey, from flower to seed to new plant, is a testament to the dandelion's remarkable adaptability and resilience.The lessons of the dandelion are applicable to our own lives. We, too, face challenges and adversity, whether it be personal struggles or external pressures. Like the dandelion, we must learn to adapt and persevere, finding the strength to grow and flourish in spite of the difficulties.The dandelion teaches us that resilience is not just about surviving but about thriving. It reminds us that every challenge is an opportunity for growth and transformation. Just as the dandelion transforms from flower to seed, we can transform our challenges into opportunities for growth and self-discovery.Furthermore, the dandelion's ability to find life in unexpected places teaches us about the importance of maintaining an open mind. Just as the seeds land in unexpected places, we should be willing to explore newhorizons and embrace opportunities that may not be immediately apparent.In conclusion, the humble dandelion is a powerfulsymbol of resilience, adaptability, and survival. Its story, though often overlooked, holds valuable lessons for us all. By emulating the dandelion's tenacity and adaptability, we can learn to navigate the challenges of life with greater ease and grace, growing stronger and more resilient with each passing day.**探索蒲公英的奥秘:一种微小却强大的自然之力** 在自然界这幅宏伟的画卷中,卑微的蒲公英常常被人们忽视,被其更为炫耀的邻居所掩盖。

中国新纪录双侨水螨亚属一新种(盾水螨总科:盾水螨科)(英文)

中国新纪录双侨水螨亚属一新种(盾水螨总科:盾水螨科)(英文)

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( 贵州 大学 昆虫研 究所 , 贵州 山地农业 病虫 害重点 实验 室 , 贵州 贵 阳 5 0 2 ) 505
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prescott微生物学 中译本

prescott微生物学 中译本

prescott微生物学中译本微生物学是研究微生物生命活动及其应用的学科。

自约翰·波曼诺·普雷斯科特教授首次出版 "Prescott's Microbiology" 以来,该教科书一直被广泛接受并被全球微生物学家所推崇。

他将微生物学体系化,并系统而全面地介绍了相关知识。

如今,该教材的中译本在中国的微生物学研究中发挥了重要作用。

Prescott微生物学中译本的出版,填补了内地微生物学教材的空白。

它不仅在帮助学生掌握基础知识方面起到了积极作用,还促进了中外学术交流,提高了国内微生物学研究水平。

该教材的编写是基于波曼诺·普雷斯科特教授对微生物学的深入研究和教学经验。

中译版本的作者也是国内顶级的微生物学专家,确保了内容的准确性和权威性。

教材的内容被分成多个篇章,每个篇章都覆盖了一个特定的主题,将相关的知识有机地组织在一起。

首先,教材介绍了微生物学的基本概念和分类学。

它详细解释了微生物的定义、特征以及不同类型的微生物(如细菌、真菌、病毒等)之间的区别和联系。

此外,教材还讲述了微生物的生活方式、生长规律以及它们在自然界中的分布和作用。

接下来,教材重点介绍了微生物的结构和功能。

学生可以了解到微生物体内的各个组织和器官,以及它们在微生物的生理代谢、遗传变异和适应性进化中的作用。

该部分还详细介绍了微生物对环境的响应和适应,以及细胞周期和复制等生物学过程。

此外,教材还涵盖了微生物学的应用领域。

它包括了微生物在农业、工业和医学中的应用。

例如,微生物在土壤改良、农作物保护、酿酒业等方面的作用。

此外,还介绍了微生物在制药、食品工业以及污水处理等领域中的应用。

教材的最后一部分是关于微生物学研究方法和实验技术的介绍。

学生可以了解到微生物学家在研究微生物时所用的常用技术和实验流程。

这部分内容对于帮助学生培养科学研究思维和实践能力非常重要。

总而言之,由波曼诺·普雷斯科特教授撰写的Prescott微生物学以及其中译本给予国内微生物学研究带来了积极影响。

microbiotaprocess包物种组成

microbiotaprocess包物种组成

标题:微生物过程中的物种组成摘要:微生物是地球上最古老的生命形式之一,它们在地球生命演化过程中发挥着重要作用。

微生物过程中的物种组成对于生态系统的稳定和功能具有重要影响。

本文将对微生物过程中的物种组成进行探讨,包括微生物的分类、多样性和功能特征等方面。

一、微生物的分类微生物是一类生命形式的统称,包括原核生物和真核生物两大类。

原核生物包括细菌和古细菌,真核生物包括原生生物和真菌。

细菌是最常见的微生物之一,具有单细胞结构,形态多样,广泛存在于各种生态环境中。

古细菌是另一类原核生物,生活在特殊环境中,例如高温、高盐度等条件下。

原生生物是真核生物的一类,通常为单细胞结构,包括原生动物和原生植物。

真菌是真核生物界中的一个重要类群,包括酵母菌、霉菌等,广泛存在于土壤、水体等环境中。

二、微生物的多样性微生物具有极大的多样性,包括不同的类群、形态和生活方式。

根据最新的研究发现,微生物的多样性远远超出人们的想象。

在不同的环境中,如土壤、水体、空气等,微生物的种类和数量都有所不同。

微生物的多样性对于维持生态系统的平衡和稳定起着至关重要的作用。

微生物的多样性也为人类提供了丰富的资源,例如制药、食品加工等领域都离不开微生物。

三、微生物的功能特征微生物具有丰富的功能特征,包括分解有机物、产生抗生素、参与生态系统的物质循环等。

其中,微生物的分解能力是其重要的功能特征之一,对有机物的降解和循环具有重要意义。

微生物还能够产生抗生素等有益物质,对人类健康和农业生产具有重要意义。

微生物还参与了生态系统中的氮循环、碳循环等重要过程,是生态系统中不可或缺的角色。

四、微生物过程中的物种组成分析微生物过程中的物种组成对于生态系统的功能和稳定具有重要影响。

通过对微生物过程中的物种组成进行分析,可以更好地理解和管理生态系统。

在不同的环境中,微生物的物种组成和丰度都有所不同。

在富集有机物的土壤中,腐化微生物的种类和丰度会较高;在高温环境中,嗜热微生物的种类和丰度会较高。

微生物学发展史上重要科学家及其成就

微生物学发展史上重要科学家及其成就
采用染色法鉴别细菌
1886
弗伦克尔(AlbertFraenkel)
发现引起肺炎的肺炎链球菌
1887
魏克塞尔包姆(A,Weichselbaum)
分离出引起脑膜炎的脑膜炎奈瑟菌(Neisseria meningitides)
1887
布鲁斯(D.Bruce)
鉴定布鲁氏杆菌为牛布鲁氏菌病的致病因子
1887
黑塞(Fannie Eilshemius Hesse)
1684
列文虎克(Anton vanLeeuwenhoek)
通过自制显微镜发现“微动体”,即细菌、原生动物等。
1688
雷迪(Francesco Redi)
实验证明腐肉上的蛆来自苍蝇的卵。
1735
林奈(Carolus Linnaeus)
建立自然分类和双名法
1748
尼达姆(John Needham)
用“干草等浸泡在烧瓶中会产生微生物”的实验证明“自然发生论”
证实梅毒(syphilis)的致病因子——梅毒螺旋体(Treponenza pallidum)
1906
沃瑟曼(August vonWasserman),奈瑟尔(A.Neisser)和 布鲁科(C.Bruck)
建立一种血清反应试验来检查梅毒,即沃瑟曼氏反应(Wasserman reaction)
1897
卡尔(R.Kraus)
发现沉淀素(precipitins)和沉淀反应
1897
毕希纳(EduardBuchner)
制备酵母浸出物进行酒精发酵
1897
爱尔里希(PaulEhrilich)
阐明抗体形成的侧链理论(1908年获诺贝尔奖)
1897
罗斯(RonaldRoss)

微生物英文词汇

微生物英文词汇

微生物英文词汇active immunity(主动免疫);active transport(主动运输);Alcohol fermentation(乙醇发酵);aerobe(好氧微生物);aflatoxin(黄曲霉毒素);AIDS(爱滋病);Ames test(艾姆氏实验);anabolism(合成代谢);anaerobe(厌氧微生物);antibiotic(抗生素);antibody(抗体);antigen(抗原);antigenic determinant(抗原决定基);antimetabolite(抗代谢物);antiseptic(防腐剂);antiserum(抗血清);antitoxin(抗毒素);arthrospore(节孢子);ascospore(子囊);asepsis(无菌);autoantibody(自身抗体);autoantigen(自身抗原);autoimmune disease(自身免疫疾病);bacteriophage(噬菌体);bacteriostatic(抑菌);binary fission(二分裂);broad spectrum(广谱);Capsid(衣壳);capsomer(衣壳粒):capsule(荚膜):Catabolism(分解代谢):cell-mediated immune(细胞介导免疫):chemoautotroph(化能自养菌):chemotaxis(趋化性):Chemotherapy(化学治疗剂):chitin(几丁质):complement(补体):Conldia(分生孢子):Conjugation(接合):Colony(菌落):Contaminant(污染物):Culture(培养物):differential medium(鉴别培养基):differential stain(鉴别染色):Disinfection(消毒):ELISA(酶联免疫):endospore(芽孢):endotoxin(内毒素):enriched medium(加富培养基):enveloped virus(包膜病毒):essential nutrient(必须营养):eucaryotic cell(真核细胞):Exotoxin(外毒素):Facultative(兼性的):Fermentation(发酵):Flagellum(鞭毛)Genotype(表型):Glycolysis(糖酵解):Gram stain(革兰氏染色):Granulocyte(粒细胞):growth factor(生长因子)Halophlle(嗜盐菌):H antigen(H-抗原):helper T cell(辅助T-细胞):Heterotroph(异养菌):Immunity(免疫):immunogen(免疫原):immune system(免疫系统):immunoglobulin(免疫球蛋白):Inclusion(内含物):Infection(感染):infectious disease(感染性疾病):Inflammation(发炎):Inoculation(接种):Interferon(干扰素):Isolation(分离):Latency(潜伏):L form(L-型菌):Lipopolysaccharide(脂多糖,LPS):Lysis(溶解):lysosome (溶酶体):病毒学virology噬菌体学bacteriophagology细菌学bacteriology鉴定细菌学determinative bacteriology系统细菌学systematic bacteriology真菌学mycology原生生物学protistology原生动物学protozoology普通微生物学general microbilogy微生物分类学microbial taxonomy微生物生理学microbial physiology微生物生物化学microbial biochemistry微生物遗传学microbial genetics微生物生态学microbial ecology古微生物学paleomicrobiology土壤微生物学soil microbiology水生微生物学aquatic microbiology海洋微生物学marine microbiology悉生生物学gnotobiology医学微生物学medical microbiology兽医微生物学veterinary microbiology农业微生物学agricultural microbiology工业微生物学industrial microbiology石油微生物学petroleum microbiology食品微生物学food microbiology乳品微生物学diary microbiology瘤胃微生物学rumen microbiology诊断微生物学diagnostic microbiology病原学etiology国际微生物学会联合会International Union of Microbiological Societies, IUMS中国微生物学会Chinese Society for Microbiology, CSM世界培养物保藏协会World Federation for Culture Collection, WFCC中国微生物菌种保藏管理委员会China Committee for Culture Collection of Microorganisms,CCCCM美国模式培养物保藏所American Type Culture Collection, A TCC自然发生说,无生源说spontaneous generation, abiogenesis原界urkingdom始祖生物progenote古始生物界archetista古细菌archaebacteria原生生物protista原生动物protozoan原生植物protophyte真核生物eukaryote原核生物prokaryote裂殖植物schizophyte微生物microorganism数值分类法numerical taxonomy模式目type order模式科type family模式属type genus模式种type species模式株type strain真菌fungi捕食真菌predacious fungi虫道真菌ambrosia fungi地下真菌hypogeal fungi虫生真菌entomogenous fungi菌根真菌mycorrhizal fungi木腐菌wood-decay fungi霉菌mold, mould半知菌imperfect fungi子囊菌ascomycetes粘菌slime mold, slime mould壶菌chytrid卵菌oomycetes接合菌zygomycetes担子菌basidiomycetes核菌pyrenomycetes盘菌cup fungi块菌truffles锈菌rust fungi蘑菇mushrooms毒蘑菇poisonous mushroom酵母菌yeast无孢子酵母菌asporogenous yeasts 有孢子酵母菌sporogenous yeasts 黑粉菌smut fungi双态性真菌dimorphic fungi毛外癣菌ectothrix毛内癣菌endothrix完全真菌perfect fungi黑粉病smut disease锈病rust disease菌丝hypha菌髓trama假菌丝体pseudomycelium气生菌丝体aerial mycelium基内菌丝体substrate mycelium球拍状菌丝体racquet mycelium 结节状菌丝nodular mycelium梳状菌丝pectinafe mycelium螺旋菌丝spiral mycelium匍匐菌丝stolon次生菌丝体secondary mycelium有隔菌丝septate hypha无隔菌丝nonseptate hypha生殖菌丝体reproductive mycelium 营养菌丝体vegetative mycelium不育菌丝体sterile mycelium菌丝体mycelium黄癣菌丝favic chandelier mycelium 产囊丝ascogenous hypha产囊体ascogonium原植体thallus粘菌体aethalium合胞体syncytium虫菌体hyphal body盾状体clypeus子实体fruiting body产孢体gleba子实层体hymenophore子实层hymenium子实下层subhymenium菌丝层subiculum菌丝段hyphal fragment菌丝束coremium菌丝索funiculus菌核sclerotium器菌核pycnosclerotium菌环annulus菌裙indusium菌盖pileus顶体apicle藏卵器oogonium雄器antheridium[锈菌]性孢子器pycnium锈子器aecium精子器spermogonium囊状体cystidium粉孢子梗oidiophore小梗sterigma接合孢子柄zygosporophore孢囊柄sporangiophore配囊柄suspensor孢子梗sporophore分生孢子梗conidiophore雄器柄androphore帚状枝penicillus瓶梗phialide梗基metulae芽孔germ pore芽管germ tube芽缝germ slit孢丝capillitium周丝periphysis类周丝periphysoid侧丝paraphysis拟侧丝pseudoparaphysis类侧丝paraphysoid[孢子]外壁exosporium外生菌根ectomycorrhiza内生菌根endomycorrhiza内外生菌根ectendomycorrhiza泡囊丛枝菌根vesicular-arbuscular mycorrhiza 刺突spike弹丝elater刚毛seta微体microbody泡囊vesicle隔膜septum假隔膜pseudoseptum分生孢子盘acervulus分生孢子座sporodochium精子团spermatium囊基膜hypothallus囊层基hypothecium囊层被epithecium囊间丝hamathecium囊托apophysis囊领collarette囊轴columella孔口ostiole菌托volva孢子角cirrus孢子球spore ball孢子印spore print聚簇cluster[菌丝]融合anastomosis[孢子]切落abjunction[孢子]缢断abstriction多态[现象] polymorphism缢缩[作用] constriction粉孢子oidium孢子spore掷孢子ballistospore厚壁孢子chlamydospore环痕孢子annellospore节孢子arthrospore卷旋孢子helicospore腊肠形孢子allantospore孔出孢子porospore星形孢子staurospore线形孢子scolecospore砖格孢子dictyospore侧生孢子aleuriospore芽生孢子blastospore瓶梗孢子phialospore无梗孢子thallospore分生孢子conidium大分生孢子macroconidium小分生孢子microconidium节分生孢子arthroconidium芽分生孢子blastoconidium器孢子pycnidiospore无隔孢子amerospore双胞孢子didymospore多隔孢子phragmospore休眠孢子hypnospore顶生孢子acrospore顶生厚壁孢子fuseau内分生孢子endoconidium担孢子basidiospore双孢担孢子dispore同形孢子isospore柄生孢子stylospore[锈菌]性孢子pycniospore产雄器孢子androspore锈孢子aeciospore夏孢子urediniospore, aeciospore 冬孢子teliospore四分孢子tetraspore粘孢子myxospore多核孢子coenospore孢囊孢子sporangiospore子囊孢子ascospore多核细胞coenocyte分生孢子果conidiocarp分生孢子器pycnidium孢[子]囊sporangium柱孢子囊merosporangium四分孢子囊tetrasporangium原孢子囊prosporangium多核孢子囊coenosporangium 休眠孢子囊hypnosporangium 子囊ascus接合孢子zygospore拟接合孢子azygospore原囊壁子囊prototunicate ascus 单囊壁子囊unitunicate ascus 双囊壁子囊bitunicate ascus子囊果ascocarp子囊壳perithecium闭囊壳cleistothecium闭囊果cleistocarp盘状子囊果discocarp孢囊果sporangiocarp[接]合子zygote单性合子azygote多核合子coenozygote异形合子heterozygote合子核zygotonucleus游动合子planozygote担子basidium半担子hemibasidium隔担子heterobasidium无隔担子holobasidium有隔担子phragmobasidium内生担子endobasidium原担子protobasidium上担子epibasidium下担子hypobasidium同担子homobasidium担子果basidiocarp担子体basidiophore配子gamete原配子progamete雄配子androgamete雄核发育androgenesis同形配子isogamete异形配子heterogamete游动配子zoogamete多核配子coenogamete配子囊gametangium配子母细胞gametocyte同形配子囊isogametangium原配子囊progametangium小孢子囊sporangiole微包囊microcyst足细胞foot cell脚胞foot cell固着器holdfast附着枝hyphopodium吸盘sucker锁状细胞clamp cell锁状联合clamp connection偶核细胞zeugite卵球oosphere卵质ooplasm孢原质sporoplasm卵配子oogamete卵孢子oospore球状胞sphaerocyst子囊腔locule子囊盘apothecium子囊座ascostroma缝裂壳hysterothecium下子座hypostroma包被peridium子座stroma壳心centrum拟包被pseudoperidium无融合生殖apomixis同宗配合homothallism准性生殖parasexuality异宗配合heterothallism同配生殖isogamy异配生殖heterogamy无配生殖apogamy配囊交配gametangial copulation 交配型mating type全型holomorph夏孢子期uredostage冬孢子堆teleutosorus, telium夏孢子堆uredinium子囊孢子形成ascosporulation孢子形成sporulation细菌bacteria薄壁[细]菌类gracilicutes硬壁[细]菌类fermicutes疵壁[细]菌类mendosicutes无壁[细]菌类tenericutes柔膜细菌mollicutes真细菌eubacteria暗细菌scotobacteria无氧光细菌anoxyphotobacteria生氧光细菌oxyphotobacteria放线菌actinomycetes螺[旋]菌spirilla粘细菌slime bacteria鞘细菌sheathed bacteria柄细菌caulobacteria弧菌vibrio根瘤细菌root nodule bacteria硫酸盐还原菌sulfate reducting bacteria硫细菌sulfur bacteria铁细菌iron bacteria紫色无硫细菌purple nonsulfur bacteria产甲烷菌methanogen硝化细菌nitrobacteria反硝化细菌denitrifying bacteria固氮细菌nitrogen fixing bacteria甲基营养菌methylotrophic bacteria产乙酸菌acetogen同型[产]乙酸细菌homoacetogenic bacteria光合作用细菌photosynthetic bacteria产氢产乙酸细菌hydrogen-producing acetogenic bacteria 同型发酵乳酸菌homofermentative lactic bacteria异型发酵乳酸菌heterofermentative lactic bacteria产氢菌hydrogenogens产气菌aerogen不产气菌anaerogen发光细菌luminous bacteria产色细菌chromogenic bacteria化能异养菌chemoheterotrophic bacteria化能自养菌chemoautotrophic bacteria光能异养菌photoheterotrophic bacteria光能自养菌photoautotrophic bacteria化能有机营养菌chemoorganotrophic bacteria 化能无机营养菌chemolithotrophic bacteria 光能有机营养菌photoorganotrophic bacteria 光能无机营养菌photolithotrophic bacteria有机营养菌organotrophic bacteria无机营养菌lithotrophic bacteria贫[营]养细菌oligotrophic bacteria一氧化碳营养菌carboxydotrophic bacteria自养菌autotrophic bacteria异养菌heterotrophic bacteria光养菌phototrophic bacteria需氧菌aerobe微需氧菌microaerobe耐氧菌aerotorelant bacteria厌氧菌anaerobe兼性厌氧菌facultative anaerobe专性厌氧菌obligate anaerobe溶原性细菌lysogenic bacteria腐生菌saprophytic bacteria苛求菌fastidious microorganism极端细菌extreme bacteria嗜压菌barophilic bacteria嗜盐菌halophilic bacteria嗜铁菌siderophilic bacteria嗜高渗细菌osmophilic bacteria微嗜氮菌oligonitrophilic bacteria嗜冷[细]菌psychrophilic bacteria嗜酸菌acidophilic bacteria嗜硫菌thiophilic bacteria中温菌mesophilic bacteria耐热细菌thermophilric bacteria氢营养菌hydrogenotrophic bacteria肠道细菌intestinal bacteria类菌体bacteroid细菌小体bacteriosome微生子gonidium蓝细菌cyanobacteria[蓝细菌]连锁体hormogonium类囊体thylakoid藻胆蛋白体phycobilisome静息孢子akinete滑行gliding异形[囊]胞heterocyst化学型chemotype化学变型chemovar血清型serotype血清变型serovar致病型pathotype致病变型pathovar生物型biotype生物变型biovar形态型morphotype形态变型morphovar革兰氏阳性菌Gram-positive bacteria 革兰氏阴性菌Gram-negative bacteria 球菌coccus双球菌diplococcus四联球菌tetrads八叠球菌sarcina球杆菌coccobacillus杆菌rod双杆菌diplobacillus棒状菌corynebacteria[细菌]毛状体trichome单鞭毛菌monotricha周[鞭]毛菌peritricha丛[鞭]毛菌lophotricha两端单[鞭]毛菌amphitrichate单端丛[鞭]毛菌cephalotricha滑行细菌gliding bacteria细菌L-型L-form of bacterium菌落colony酵母型菌落yeast type colony类酵母型菌落yeast like colony次生菌落secondary colony粗糙型菌落rough colony光滑型菌落smooth colony丝状型菌落filamentous type colony 子菌落daughter colony深层菌落deep colony粘液型菌落mucoid colony巨大菌落giant colony侏儒型菌落dwarf colony菌苔lawn菌胶团zoogloea菌膜pellicle[菌]醭mycoderm, pellicle群游现象swarming菌柄stipe[菌体]附器appendage鞭毛flagellum周质鞭毛periplasmic flagella轴丝axial filament菌毛pilus性丝sex pilus外生孢子exospore内生孢子endospore芽孢spore芽孢形成sporulation终端芽孢terminal spore近端芽孢subterminal spore中生芽孢central spore前芽孢forespore[芽孢]皮层cortex芽孢外膜exitine芽孢内膜intine外壁exine伴胞晶体parasporal crystal菌蜕ghost鞘sheath荚膜capsule粘液层slime layer微荚膜microcapsule壁膜间隙periplasmic space原生质体protoplast原生质球spheroplast气泡gas vacuole甲烷粒体methanochondria间体mesosome载色体chromatophore鞭毛基体flagellar basal body异染质volutin异染粒matachromatic granules致死颗粒killer particle紫膜purple membrane噬菌体bacteriophage无囊盖类inoperculatae超显微微生物ultramicroscopic organism 真菌噬菌体mycophage噬藻体phycophage烈性噬菌体virulent phage温和噬菌体temperate phage前原噬菌体preprophage原噬菌体prophage隐性前噬菌体cryptic prophage营养期噬菌体vegetative phage载体噬菌体carrier phageλ噬菌体lambda particles phage [可]诱导噬菌体inducible phage同源免疫噬菌体homoimmune phage 噬菌体分型bacteriophage typing噬菌体型phagetype噬菌体变型phagevar噬斑plaque[噬菌体]聚合头部polyhead[噬菌体]聚合尾鞘polysheath[噬菌体]伞毛fimbrium[噬菌体]颈须whisker[噬菌体]先导蛋白pilot protein[噬菌体]尾丝抗原fiber antigen[噬菌体]顶体apex[噬菌体]基片插孔base-plate hub [噬菌体]基片丝base-plate fibril [噬菌体]基片楔突base-plate wedge [噬菌体]串联体concatemer[噬菌体]颈部collar[噬菌体]顶部壳粒apical capsomere [噬菌体]尾丝tail fiber[噬菌体]畸形体monster[噬菌体]颈圈connector[噬菌体]髓部core[噬菌体]头部head[噬菌体]尾部tail[噬菌体]尾管tail tube[噬菌体]尾鞘tail sheath类病毒viroid病毒virus真病毒euvirus亚病毒subvirus原病毒provirus拟病毒virusoid卫星病毒satellite virus假型病毒pseudotype virus慢病毒slow virus辅助病毒helper virus过客病毒passenger virus多分体病毒multicomponent virus昆虫痘病毒entomopox virus, EPV颗粒体症病毒granulosis virus, GV多角体病毒polyhedrosis virus核型多角体病毒nuclear polyhedrosis virus, NPV质型多角体病毒cytoplasmic polyhedrosis virus,CPV 多粒包埋型病毒multiple embedded virus单粒包埋型病毒singly embedded virus伴随病毒associated virus浓核病毒densovirus,DNV内源病毒endogenous virus潜伏病毒latent virus肠道病毒enterovirus艾柯病毒ECHO virus虫媒病毒arbovirus腺病毒adenovirus腺伴随病毒adeno associated virus真菌病毒mycovirus肿瘤病毒oncovirus逆[转]录病毒retro virus坏死病毒necrosis virus虹彩病毒irido virus泛嗜性病毒pantropic virus毒株strain原[生小]体elementary body包含体inclusion body顾氏小体Guarnieri's bodies内氏小体Negri's body病毒[粒]体virion裸露病毒[粒]体naked virion假病毒体pseudovirion立体对称cubical symmetry二十面体对称icosahedral symmetry螺旋对称helical symmetry[病毒]五邻体pentomer,pentons[病毒]六邻体hexonmer,hexons复合对称complex symmetry包膜突起peplomerbody包膜envelope, peplos蛋白质包膜protein envelope[病毒]包膜抗原envelope antigen[病毒]壳体capsid[病毒]壳粒capsomer, capsomere二十面[体]壳体icosahedron capsid 核心core核壳nucleocapsid病毒原质体viroplasma病毒束virus bundle多角体polyhedron多角体蛋白polyhedrin颗粒体granule颗粒体蛋白granulin类核nucleoid内含颗粒inclusion granuleX体X-body[病毒]早期蛋白early protein[病毒]晚期蛋白late protein负链negative strand正链positive strand复制子replicon病毒发生基质virogenic stroma衣原体chlamydia[衣原体]始体initial body立克次氏体rickettsia假肽聚糖pseudopeptidoglycan肽聚糖peptidoglycan磷壁酸teichoic acid胞壁酸muramic acid2,6-吡啶二羧酸dipicolinic acid, DPA 脂多糖类lipopolysaccharides多糖包被glycocalyx鞭毛蛋白flagellin菌毛蛋白pilin杀白细胞素leucocidin豆血红蛋白leghaemoglobin藻胆蛋白phycobiliprotein藻青蛋白phycocyanin藻红蛋白phycoerythrin藻青素cyanophycin藻蓝素algocyan, leucocyan藻胆素phycobilin藻红[胆]素phycoerythrobilin藻蓝胆素phycocyanobilin藻青素颗粒cyanophycin granule别藻蓝素allophycocyanin类葫萝卜素carotenoids细菌淀粉粒granulose聚β羟基丁酸盐poly-β-hydroxy butyrate葡萄球菌A蛋白staphylococcal protein A, SPA 纯化蛋白衍生物purified protein derivative, PPD [葡萄球菌]凝固酶staphylocoagulaseβ[细胞]溶素β-lysinα淀粉酶α-amylase通透酶permease胞内酶intracellular enzyme胞外酶extracellular enzyme果胶酶pectinase逆[转]录酶reverse transcriptase凝固酶coagulase受体破坏酶receptor destroying enzyme, RDE透明质酸酶hyaluronidase纤维素酶cellulase链道酶streptodornase,SD链激酶streptokinase,SK神经氨酸酶neuraminidase青霉素酶penicillinase溶菌酶lysozyme[细菌]紫膜质bacteriorhodopsin菌紫素bacteriopurpurin[细]菌[叶]绿素bacteriochlorophyll自溶素autolysin亲菌素bacteriotropin攻击素aggressin抑殖素ablastin粘附素adhesin菌红素bacterioerythrin灵菌毒素prodigiosus toxin细菌素bacteriocin麻风菌素lepromin葡萄球菌素staphylococcin伞菌氨酸agarfitine苏云金菌素thuricin肠球菌素enterococcin布氏菌素brucellin大肠菌素colicin, colicine丁香假单胞菌素syringacin黄色粘球菌素xanthacin链球菌素streptocin流产菌素abortin绿脓[菌]素pyocyanin红假单胞菌素rhodopseudomonacin 绿脓菌荧光素pyofluorescein白喉毒素diphtheria toxin杯伞素clitocybine白细胞溶素leucolysin表皮溶解毒素epidermolytic toxin 产气荚膜梭菌素perfringocin肠毒素enterotoxin毒蝇碱muscarine肺炎球菌毒素pneumotoxin鬼笔[毒]环肽phalloidin根霉蝶呤rhizopterin肺炎[链]球菌溶血素pneumolysin 黑粉菌酸ustilagic acid分枝菌酸mycolic acid齿孔酸eburicoic acid根霉促进素rhizopin蘑菇素agaricin蘑菇酸agaricinic acid红斑毒素erythrogenic toxin黄曲霉毒素aflatoxin菌丝酰胺mycelianamide绿脓杆菌溶血素pyocyanolysin葡萄球菌溶血毒素staphylolysin真菌毒素mycotoxin曲霉毒素aspertoxin赭曲毒素ochratoxin曲酸kojic acid破伤风[菌]痉挛毒素tetanospasmin 溶葡萄球菌素lysostaphin破伤风[菌]溶血素tetanolysin溶纤维蛋白溶酶fibrinolysin溶血素hemolysin鼠疫菌素pesticin神经毒素neurotoxin杀[细]菌素bactericidin外毒素exotoxin内毒素endotoxin细菌毒素bacteriotoxin血凝素hemagglutinin杂色曲霉素A versicolorin A柄曲霉素sterigmatocystin毒植物素phytotoxin真菌醇mykol链球菌溶血素streptolysin剥脱性毒素exfoliative toxin细菌荧光素bacteriofluorescein[放线菌]土臭味素geosmins土壤杆菌素agrobacteriocin产甲烷[作用] methanogenesis生物转化bioconversion生长因子growth factor420 因子factor 420V 因子V factorX 因子X factormixed culture(混合培养):monoclonal antibody(单克隆抗体):Monocyte(单核细胞):Mutagen(诱变剂):Mutation(突变)Mycelium(菌丝体):narrow spectrum(窄谱):negative stain(负染色):nitrogen fixation(固氮):Nucleocapsid(核衣壳):Nucleoid(拟核):Nutrient(营养物质):Obligate(专性的):Parasite(寄生):Pasteurization(巴斯德消毒):Pathogen(病原体):Saprophytes(腐生型)Pathogenidty(致病性):Pathology(病原学):passive transport(被动扩散);Penicillins(青霉素):Peptidoglycan(肽聚糖):Plasmids(质粒)periplasmic space(周质空间):Phage(噬菌体):Phenotype(表型):Photoautotroph(光能自养菌):Pilus(性丝);prophage(前噬菌体):Protoplast(原生质体):Pseudohypha(假菌丝):Psychrophile(嗜冷菌):respiratory chain(呼吸链):reverse transcriptase(逆转录酶):SCP(单细胞蛋白):selective media(选择培养基):Serotyping(血清型):sexual reproduction(有性繁殖)Spheroplast(球形体):spike(刺突):Spirillum(螺菌):Spirochete(螺旋体):Sporangium(孢囊):Sterilization(灭菌):A Strain(菌株):subcellular vaccine(亚单位疫苗):superoxide ion(超氧离子):suppressor T cell(抑制T细胞):temperate phage(温和噬菌体):thermal death point(致死温度):thermal death time( 热致死时间):Therrnophlle(嗜热菌):Toxoid(类毒素):Transduction(转导):Transformation(转化):Transposon(转座):V accine(免疫法):V irold(类病毒):Zygospore(接合孢子)。

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Discovery of Porcine microRNAs and Profiling from Skeletal Muscle Tissues during DevelopmentTing-Hua Huang,Meng-Jin Zhu,Xin-Yun Li,Shu-Hong Zhao*Key Laboratory of Agricultural Animal Genetics,Breeding,and Reproduction of Ministry of Education&Key Laboratory of Swine Genetics and Breeding of Ministry of Agriculture,Huazhong Agricultural University,Wuhan,People’s Republic of ChinaAbstractMiRNAs(microRNAs)play critical roles in many important biological processes such as growth and development in mammals.In this study,we identified hundreds of porcine miRNA candidates through in silico prediction and analyzed their expression in developing skeletal muscle using microarray.Microarray screening using RNA samples prepared from a33-day whole embryo and an extra embryo membrane validated296of the predicted parative expression profiling across samples of longissimus muscle collected from33-day and65-day post-gestation fetuses,as well as adult pigs, identified140differentially expressed miRNAs amongst the age groups investigated.The differentially expressed miRNAs showed seven distinctive types of expression patterns,suggesting possible involvement in certain biological processes.Five of the differentially expressed miRNAs were validated using real-time PCR.In silico analysis of the miRNA-mRNA interaction sites suggested that the potential mRNA targets of the differentially expressed miRNAs may play important roles in muscle growth and development.Citation:Huang T-H,Zhu M-J,Li X-Y,Zhao S-H(2008)Discovery of Porcine microRNAs and Profiling from Skeletal Muscle Tissues during Development.PLoS ONE3(9):e3225.doi:10.1371/journal.pone.0003225Editor:Suzannah Rutherford,Fred Hutchinson Cancer Research Center,United States of AmericaReceived March24,2008;Accepted August15,2008;Published September16,2008Copyright:ß2008Huang et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.Funding:Financial support was provided by the Key Project of National Basic Research and Developmental Plan(2006CB102105)and the National Natural Science Foundation(30671138)of China.Competing Interests:The authors have declared that no competing interests exist.*E-mail:shzhao@IntroductionThe recent discovery of miRNAs introduced a new mechanism of gene expression regulation[1,2].Despite the fact that biological functions have been assigned to only a few dozen miRNAs,it is becoming apparent that miRNAs participate in the regulation of a variety of developmental and physiological processes[3].Not surprisingly,recent studies have shown that miRNAs play important roles in the regulation of muscle development.The functional characterization of miR-1and miR-133has been an important step in our understanding of miRNA-mediated muscle development.miR-1-1and miR-1-2were first found to be specifically expressed in mouse cardiac and skeletal muscle precursor cells and were found to be transcriptionally regulated by the myogenic differentiation factors MyoD,Mef2,and SRF[4]. Overexpression of miRNA-1in the mouse developing heart has a negative effect on muscle proliferation as it targets the transcrip-tion factor that promotes ventricular cardiomyocyte expansion, Hand2[4].In Drosophila,the expression of miR-1is controlled by the Twist and Mef2transcription factors[5].Investigation of a loss-of-function phenotype of Drosophila miR-1showed that miR-1 is not required for the formation or physiological function of the larval musculature,but is required for the post-mitotic growth of larval muscle[5].Recent studies showed that miR-1promotes myogenesis by targeting histone deacetylase4(HDAC4),a transcriptional repressor of muscle gene expression,and that miR-133enhances myoblast proliferation by repressing serum response factor(SRF)[6],both examples of new molecular mechanisms to regulate skeletal muscle gene expression and embryonic developmental[6].Another miRNA,miR-206,has also been characterized as a muscle regulator in recent studies.In co-operation with miR-133, miR-206can repress myoblast fusion by targeting the connexin43 (Cx43)gap junction channels without altering the Cx43mRNA level[7].These findings have generated more detailed insights into the mechanisms underlying the myogenesis process and have uncovered different pathways that lead to myofiber proliferation and differentiation.However,the complete roles of miRNAs in muscle growth&development still remain to be elucidated.In mammals,muscle mass is mainly determined by the number and size of muscle fibers.In the pig,for example,the number of muscle fibers is prenatally determined during primary and secondary muscle fiber formation,while the postnatal hypertrophy process then increases the length and diameter of these fibers. Primary muscle fiber formation begins at approximately30days following gestation.Secondary muscle fiber formation begins at about50to60days post-gestation,when myoblasts align and fuse to form secondary muscle fibers at the surface of existing primary muscle fibers[4].Identification of genes governing these processes will provide insights into the regulation of muscle growth. Currently,numerous genes,including growth factors,regulatory proteins,receptors,and transcription factors have been identified as participating in the regulation of the myogenesis.However,the underlying molecular pathway elements,such as the decisive secondary regulatory factors of the major genes responsible for controlling prenatal muscle growth,remains poorly understood. We hypothesized that there were more miRNAs associated with muscle growth and development in prenatal pigs yet to be discovered.Profiling of transcriptome changes of mature miRNAsisolated from key developmental stages is a promising technique to use in uncovering these miRNAs.In the present study,we identify miRNAs whose expression has not previously been reported in pigs.Our results also identify a number of differentially expressed miRNAs that could represent new regulatory elements in muscle growth and development.Results and DiscussionIdentification of porcine miRNA candidatesIn silico porcine miRNA prediction by homolog searches.We made use of the property of miRNAs to be highly conserved between closely related species in order to predict novel porcine miRNA candidates[8].Pair-wise comparison of the porcine genomic sequences(August2007)to hairpin sequences collected from mirBase(Version10.0)resulted in12,048alignments. After removing the redundant alignments,we ended up with a total of775unique porcine miRNA candidates(Table S1).All candidates were found to have the potential for the hairpin-loop secondary structures typical to known miRNA transcripts.Among these candidates,49had been reported while the remaining were new. Homolog search and de novo prediction are two typical approaches widely used in miRNA prediction.The homolog search approach is essential in our study since the porcine genome is not yet available for a direct prediction.Although the approach is limited by its inability to detect less conserved miRNAs,it is a nonetheless efficient and cost-effective.Detection of expressed porcine miRNAs by microarray hybridization.To validate these miRNA candidates,a recently developed mammalian miRNA microarray was used to evaluate the expression of porcine miRNAs.At the design time of the microarray, there were576human miRNAs,238rat miRNAs and358mouse miRNAs reported.After removing the redundant sequences,there remained743unique mature miRNA sequences.The microarray was designed to contain743probes complementary to these sequences(See probe list of the microarray in Table S2).The in silico prediction mentioned above was based on the alignment of the reported miRNAs of human,mouse and rat to the porcine genomic sequences.As expected,the microarray covered all of the candidates found by this method,and thus can be used to detect their expression. Microarray hybridization with RNA samples prepared from the 33-day post-gestation stage porcine whole embryo(E33.f)and placenta(P33.p)detected expression of296miRNAs(230in E33.f and275in E33.p,signal.2Mean+2SD.See full list in Table S3.1). For the49porcine miRNAs deposited in miRbase,41of them were detected(35in E33.f and39in E33.p).The six porcine miRNAs identified by Kim et al.were also detected[9].The remaining255 miRNAs have not been previously reported to be expressed in pig. We also found a large number of probes that showed strong signals but were not included in our candidate list,such as the miR-13and miR-557.The failure to detect these candidates by the homolog search method is possibly due to the fact that only part of the porcine genome(60%)was available at the time.The first reported porcine miRNA was the identification of the mir17-92cluster using the homolog search method[10].A more extensive homology search has since been performed by Kim et al.[9].They identified58candidates and validated six of them by northern blot.Other miRNA entries in miRBase are predictions found by genomic comparisons with other model organisms such as human,mouse and rat without proof of expression[11].There are49miRNAs reported so far.Our experiments expanded the number of porcine miRNAs(with identified sequence and confirmed expression)to116(Table1lists the top20highlyTable1.New porcine miRNAs identified in33day post-gestation samples of whole embryo(E33.f)and placenta(E33.p).MiRNA Name Microarray Probe Sequence Porcine Trace Sequence Normalized Expression LevelE33.p E33.fssc-let-7d ACTATGCAACCTACTACCTCT gnl|ti|13808200927274.3325372.67ssc-let-7e ACTATACAACCTCCTACCTCA gnl|ti|157774834616353.3318411.33ssc-mir-10b ACAAATTCGGTTCTACAGGGTA gnl|ti|202273040616918.677983.33ssc-mir-124a-1TGGCATTCACCGCGTGCCTTAA gnl|ti|142067012121323373ssc-mir-15b TGTAAACCATGATGTGCTGCTA gnl|ti|2020963538936112985ssc-mir-16-1CGCCAATATTTACGTGCTGCTA gnl|ti|157997182122503.3326688ssc-mir-17ACTACCTGCACTGTAAGCACTTTG gnl|ti|157990983232828.679736.67ssc-mir-191AGCTGCTTTTGGGATTCCGTTG gnl|ti|20253940359408.3314004.33ssc-mir-199(a/b)AACCAATGTGCAGACTACTGTA gnl|ti|(2019854499/1377265104)40134.6742470ssc-mir-19b-1TCAGTTTTGCATGGATTTGCACA gnl|ti|157990983830333.338530ssc-mir-22ACAGTTCTTCAACTGGCAGCTT gnl|ti|13776390705877.3327348.67ssc-mir-29a AACCGATTTCAGATGGTGCTA gnl|ti|8606095555892.336809.67ssc-mir-30b AGCTGAGTGTAGGATGTTTACA gnl|ti|1574275341695817706.67ssc-mir-30d CTTCCAGTCGGGGATGTTTACA gnl|ti|100861700314482.6719839.33ssc-mir-320TTCGCCCTCTCAACCCAGCTTTT gnl|ti|20279856919252.6721585.67ssc-mir-376a-1ACGTGGATTTTCCTCTATGAT gnl|ti|10086377821147310293ssc-mir-382CGAATCCACCACGAACAACTTC gnl|ti|7755967953858.3310386.33ssc-mir-487b AAGTGGATGACCCTGTACGATT gnl|ti|8516193037105.6713520ssc-mir-99a CACAAGATCGGATCTACGGGTT gnl|ti|20209605853348835344ssc-mir-185GAACTGCCTTTCTCTCCA gnl|ti|15753678216678.6710275.67doi:10.1371/journal.pone.0003225.t001expressed new miRNAs.See the full list in Table S4.1and the predicted secondary structures in Table S4.2).Global miRNA expression profiling of porcine skeletal muscle tissuesAn overview of the expression profile.To identify themiRNAs that might be involved in muscle development and to discriminate these from the miRNAs possibly involved in promoting or repressing muscle myogenesis and differentiation,we carried out a comparative miRNA expression profile across skeletal muscle samples collected from pigs of 33-days post-gestation (E33),65-days post-gestation (E65)and adult age (Adu).Samples from each age group were collected independently and the analysis performed in triplicate to ensure parisons between each of the replicates showed that the replicates have good reproducibility (Figure 1).The use of short RNA probes antisense to the mature miRNA sequence has not proven to be an effective approach to reliably quantify the expression differences between miRNAs that have only one mismatch or a few mismatches [12].Luo et al.previously performed a sensitivity test of the microarray using the artificially transcribed miRNA of let-7a to hybridize to the let-7probe set (let-7a to let-7g,let7-i).Their results showed that the microarrayutilized in this study was able to distinguish between the mismatched sequences,but was unable to distinguish between the highly similar sequences [13].Therefore microarray results for closely related miRNAs should be interpreted with caution,as expression differences of a given miRNA could be exaggerated or diminished by the expression of their paralogs.Of the 576miRNAs on the microarray,256(44%)were expressed in the muscle samples.Of those expressed,227were in E33and 228in E65,while only 163were expressed in Adu (see Table S3.2).Taking into account the fact that miRNAs are negative regulators of coding genes that act by either inhibiting translation or inducing mRNA degradation of the target gene [3,14,15],these results suggest lower expression levels of the coding genes regulated by the miRNAs in the prenatal stages.The modulation of muscle development processes is triggered by sequential events of gene activation and inhibition.The differences in miRNA expression between the ages detected in this study support the complexity of their roles in muscle development.Differentially expressed miRNAs detected by the microarray.Of the 256miRNAs detected by the microarray,expression levels of 140of them changed significantly between the developmental stages investigated (Fold change .2,p ,0.001,FDR ,0.001,see Table S5)and 51changed more thanten-foldFigure 1.Reproducibility of the microarray experiments.We examined the miRNA expression in three developmental stages of skeletal muscle (E33,E65and Adu).Samples from each stage were isolated in triplicate and hybridized to the microarray.Scatter plots demonstrate the pair-wise comparison between each two sets of triplicates.The R represents the Spearman correlation coefficient.doi:10.1371/journal.pone.0003225.g001(Table2).For example,the average increase of miR-486signal from E33to E65was3.3-fold,and13.4-fold from E65to Adu;the average increase of miR-376b signal from E33to E65was4.6-fold, but decreased54.7-fold from E65to Adu,and therefore in Adu it appeared11.9-fold lower than in E33;miR-422a signal increased more than6.9-fold from E33to E65,after which it remained stable;miR-495signal was strong in E33and E65,but nearly undetectable in the Adu stage.Interestingly,we found that three miRNAs(miR-363,miR-365and miR-422b)were differentially expressed between E33and Adu,despite their expression not being significantly different when comparing either E33to E65or E65to Adu.This may represent a type of long term regulation. Pair-wise comparisons showed that large numbers of miRNAs are differentially expressed between any given two ages.In addition,the number of differentially expressed miRNAs as well as the value of the average fold changes varied between the three developmental ages investigated.As shown in Table3,the number of differentially expressed miRNAs between E33and E65is much smaller than between E65and Adu,and the value of the average fold change between E33and Adu is much lower than between E65and Adu.These findings show that the expression patterns of the three ages are unique.Of the three miRNAs reported as regulators of development in skeletal and cardiac muscle,miR-206was found to be up-regulated2.9-fold in Adu compared to E65,but the expression variance of miR-1and miR-133failed to reach statistically significant levels.These two miRNAs showed a high level of expression in the microarray analysis,thus technical error could be ruled out.It should be noted that the functional discovery of these miRNAs was made mostly in cell culture systems,which may differ from the in vivo system.Several of the differentially expressed miRNAs identified here were shown to play roles in growth and development related processes in recent studies.These include miR-214,miR-140, miR-150,miR-10,as well as miR-181.In the zebrafish,miR-214 can modulate hedgehog signaling,thus changing muscle cell fate [18],and miR-10was shown to represses HoxB1a and HoxB3a, which are involved in patterning the anterior-posterior axis[19]. In mouse cells,the cartilage specific miRNA,miR-140,targets the histone deacetylase4(HDAC4),suggestive of a role in long bone development[20].In mature B and T cells,the miR-150was found to block early B cell development when expressed prematurely,and also found to control B cell differentiation by targeting the transcription factor of c-Myb[21].Furthermore, miR-181was found to be involved in the process of mammalian skeletal-muscle differentiation,by targeting the homeobox protein Hox-A11during mammalian myoblast differentiation[22].These findings suggest that identifying differentially expressed miRNAs may lead to the discovery of miRNAs related to muscle growth and development.Validation of the microarray results by direct quantification.Five representative differentially expressed miRNAs(miR-150,miR-193b,miR-196a,miR-187b and miR-495)were chosen for validation by the stem–loop RT–PCR basedTable2.MiRNAs differentially expressed between E33,E65and Adu stages(Fold change.10.0,p,0.001and FDR,0.001).MiRNA Name E65/E33Adu/E65Adu/E33MiRNA Name E65/E33Adu/E65Adu/E33 miR-214-0.130.06miR-493 4.620.08-miR-422a 6.93-15.01miR-409-5p 2.350.080.19miR-503-0.120.07miR-379-0.090.21miR-497- 5.8011.09miR-95-13.2316.38miR-721- 5.0011.07miR-369-5p 2.850.060.16miR-189-7.7910.37miR-557-30.9723.00miR-378- 6.2411.58miR-655-0.06-miR-487a 2.92 5.3415.59miR-656-0.08-miR-680- 4.9814.45miR-1820.06-0.13miR-127-0.030.04miR-376a 4.650.060.26miR-495-0.010.02miR-365--14.39miR-411-0.010.04miR-486 3.2613.4443.75miR-487b 4.410.04-miR-323-0.060.09miR-29a 1.9216.7832.24miR-660 2.270.080.17miR-193b-22.4122.68miR-409-3p-0.06-miR-29b-15.4932.19MIR-202 3.740.030.13miR-376b 4.590.020.08miR-382-0.030.11miR-29c-17.4417.45miR-503-0.090.07miR-376a 3.200.030.10miR-431 3.900.040.15miR-335-0.050.08miR-410-0.09-miR-411 3.280.030.09miR-150-20.7448.39miR-532-0.05-miR-380-3p-0.060.19miR-299-5p 4.730.05-miR-432 3.700.07-miR-362-0.090.13miR-196a0.07 6.10-miR-455-3p-0.08-miR-329 6.350.030.19doi:10.1371/journal.pone.0003225.t002real-time PCR method[6]using three independent samples(The primer sequences are available in Table S6).The expression levels for miR-150,miR-193b,miR-187b and miR-196a,as determined by RT-PCR,were in concordance with the normalized microarray data(Pearson correlation coefficient.0.9,q value,0.001,Figure2).In general,the results of qPCR validated the microarray results.An exception was miR-495,for which the expression levels in E33and E65varied dramatically. Although we have not verified the exact cause,the variance may come from biological differences between the samples. Furthermore,it should be noted that the purification process of the stem–loop RT–PCR assay is unable to completely remove long RNA nucleotides,thus we cannot exclude the possibility that the precursors are also quantified[6].Distinctive miRNA expression patterns during muscle development.To visually illustrate the expression type of the miRNAs being expressed during different developmental stages,a hierarchical cluster analysis was performed for the differentially expressed miRNAs.The results show that the miRNA expression patterns fall into seven typical categories:A)prenatally expressed, expression level increased between E33and E65;B)universally expressed,expression level decreased between E33and E65;C)universally expressed,expression level increased through the three ages;D)moderately expressed in E65,expression levels in E33and Adu nearly undetectable;E)moderately expressed in E33, expression levels in E65and Adu nearly undetectable;F) postnatally expressed,expression nearly undetectable in prenatal ages;G)moderately expressed,expression level increase through the three ages.The expression patterns described above are clearly reflected by the formation of several big clusters in the tree map of the clustering results(Figure3).The myogenesis process takes place mostly in the prenatal stage and becomes almost inhibited in the postnatal stage[16].It has been demonstrated in the pig that primary muscle fiber formation begins at approximately30days post-gestation and the secondary muscle fiber formation begins at about50to60days post-gestation [17].The categories of miRNA expression patterns described above provide a sensible basis for generating specific hypotheses of how miRNAs function in the biological context of the develop-mental ages investigated.The prenatally expressed miRNA clusters may include miRNAs that play roles in the promotion of myogenesis(Figure3A and B).In contrast,the postnatally expressed miRNAs clusters may include miRNAs that act as inhibitors of myogenesis(Figure3C and F).The E33and E65 highly expressed miRNA clusters may include miRNAs that play roles in the process of primary and secondary muscle fiber formation,respectively(Figure3E and D).In previous studies,we found that although the secondary muscle fiber formation took place later than the primary muscle fiber formation process,the two temporally overlapped at the beginning of secondary muscle fiber formation[17].This may be the primary reason why a large number of miRNAs are expressed both at the E33stage and the E65stage(Figure3A,B and F).Differentially expressed miRNAs may play important roles in porcine muscular development.A major challenge to understanding the function of these developmentally regulated miRNAs is the question of target identification.It is commonly recognized that the miRNA and its targets must be located in the same subcellular position to make the biological interactions operable,thus the spatial and temporal information of miRNA expression may narrow the search for miRNA target candidates. The differentially expressed miRNAs detected by this micro-array analysis provide a valuable candidate list of muscle growth and development related miRNAs.In this analysis,we used a well established miRNA-target dataset generated by TargetScan to investigate the possible functions of these miRNAs and to provide evidence for their involvement in the muscle development processFigure2.Validation of the microarray results using Real-time PCR method.Expression levels of five miRNAs(miR-150,miR-193b, miR-196a,miR-187b and miR-495)were detected by Real time PCR (right)and microarray(left).We have made a scaling to the raw data of Real time PCR to make it comparable to the microarray data.R represents the Pearson correlation coefficient.doi:10.1371/journal.pone.0003225.g002Figure3.Hierarchical cluster analysis.We performed a data adjustment(median center and normalization)in the cluster analysis.The color codes of red,white,black and dark green represents expression levels of high,average,low and absent respectively.A detailed view of the miRNA expression levels in clustering patterns is shown in the plot areas from A to G.doi:10.1371/journal.pone.0003225.g003[23,24].The relevant mRNA sequences used in this analysis are orthologous genes.In addition,the target sites were characterized as evolutionarily conserved in five species(human,mouse,rat,dog and chicken),a criterion that also acted as a good filter for false positive assignments of miRNAs to genes[24,25].Altogether we analyzed6,654genes that have at least one predicted miRNA binding site in their39UTR,and a total of84miRNA families in the TargetScan datasets.As a result,we obtained a total of24,555 predicted miRNA-mRNA interaction sites(Table S7).As we expected,most of the miRNAs investigated targeted hundreds of genes and over65%of the targets were regulated by more than one miRNA(Tables S8.1and S8.2).The high degree of connectivity between the miRNA-mRNA pairs supports the idea that these miRNAs have extensive and complicated roles during the muscle development process.Three genes,NFIB,TNRC6B and ZNF148assigned the highest number of miRNA interaction sites.The NFIB gene was previously identified as an activator of the differentiation-specific genes,such as MCSFR[26].TNRC6B was co-purified with a cytoplasmic HeLa cell protein complex containing AGO2,DICER,and MOV10,and thus is implicated in mediating miRNA-guided mRNA cleavage in HeLa cells[27]. ZNF148(alias ZBP89)was originally reported as a gastrin gene expression repressor[28,29]and recently,studies of mice expressing only ZBP89-delta-N showed significant growth delay and a reduction of viability[30].GO terms and KEGG pathway annotation of the miRNA targets using the DAVID gene annotation tool(/)further illustrate the possible roles and mechanisms of these differentially expressed miRNAs in muscle development(Document S1).The above analyses provide an overview investigation on the possible functions of differentially expressed miRNAs based on computationally predicted target datasets.Although the accuracy of the computational approaches for identification of mammalian miRNA targets is still limited[33],these results will definitely advance the hypothesis-driven functional studies of these miRNAs. Materials and MethodsHomolog search for miRNA candidatesAnalysis of the current porcine genomic draft sequences(August 2007)was performed by comparing porcine genomic sequences with both experimentally confirmed and predicted data sets from other species using BLAST(Basic Local Alignment Search Tool). The alignments,requiring at least90%pre-miRNA similarity and 100%mature miRNA similarity,were reserved for further study. The predicted miRNA secondary structure was generated by the RNAfold software package(http://www.tbi.univie.ac.at/,ivo/ RNA/RNAfold.html).We also checked the phylogenetic shadow-ing profile of these sequence pairs as characterized by the miRNA coding arm,which suffered the highest selective pressure,and then in succession the non-coding arm,the stem region,the loop region,and the flanking sequence.The candidates not following these rules were removed from the datasets.After these steps,we ended up with hundreds of miRNA candidates.Samples and RNA preparationOur experiments included three RNA samples isolated from three independent fetal or adult pigs.Sample collection was approved by the ethics committee of Huazhong agricultural university.The longissimus tissues were dissected after removing the epimysium coverings.These samples were snap-frozen in liquid nitrogen and stored at280u C.Total RNA was isolated using a Trizol protocol(Invitrogen).After quantification,the RNA was isolated using PEG(polyethylene glycol)and labeled by RNA ligase according to the method of Thomson et al.[34]. Microarray hybridization and data analysisThe microarrays used in this study were bought from CapitalBio Company(NO.225011).The hybridization was done by the CapitalBio Company service.In brief,labeled RNA was dissolved in 16ul hybridization mixture(15%formamide;0.2%SDS;36SSC; 506Denhardt’s)and hybridized overnight.The slides were washed in0.2%SDS,26SSC for four minutes at42u C,and in0.26SSC for four minutes.The slides were scanned using the LuxScan10K/A scanner(CapitalBio Company)and the raw pixel intensities were extracted using the LuxScan3.0software(CapitalBio Company). The median pixel intensities were background subtracted.Hybrid-ization signals that failed to exceed the average background value by more than two standard deviations(Signal.Mean+2SD)were excluded from analysis.In all of the three duplicate slides,probe signal.Mean+2SD was classified as detected(for E33.p and E33.f, no duplicate experiments were performed,thus signal exceeding 26Mean+2SD were defined as detected.).The data were normalized between slides from different ages groups using the quantile normalization method proposed by Bolstad et al[35].The differentially expressed genes,classified as those with Fold changes.2,P value,0.001and FDR,0.001,were selected using the SAM software,version2.1(Significance Analysis of Micro-arrays,/˜tibs/SAM/).The subsequent analysis of miRNA targets prediction and target gene functional annotation was performed using the TargetScan software(http:// /)and the DAVID gene annotation tool (/),respectively.Stem-Loop Real-time RT-PCRA miRNA quantification method similar to that described by Chen et al.[6]was used to validate the microarray data.Three independent samples from each time point were analyzed.In brief, the assay was performed using Stem–loop RT followed by SYBR Green Real-time PCR analysis.Firstly,1m g total RNA was reverse transcribed using200U M-MLV Reverse Transcriptase (Takara:02640A)and1m l Stem-loop RT primer in an Applied Biosystems9700Thermocycler with incubation at30u C for 15min,42u C for60min and85u C for5min.Importantly,all reverse transcriptase reactions were run along with‘‘no-template controls’’.The no-template controls gave non-detectable signals in all samples,confirming the high specificity of the miRNA quantification assay.Real-time PCR was performed using a standard SYBR Green PCR kit(Toyobo:QPK-201)on the BIO-RAD iQ5Real-Time PCR Detection System.Porcine Met-tRNA was used as an internal control and all reactions were run in triplicate.The DD Ct method was used to determine the expression level differences between surveyed stages[36].The significant level was set to0.05.Supporting InformationDocument S1Found at:doi:10.1371/journal.pone.0003225.s001(0.08MB DOC)Table S1Found at:doi:10.1371/journal.pone.0003225.s002(0.16MB XLS)Table S2Found at:doi:10.1371/journal.pone.0003225.s003(0.12MB XLS)。

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