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A discovery of novel microRNAs in the silkworm (Bombyx mori) genome

A discovery of novel microRNAs in the silkworm (Bombyx mori )genome

Xiaomin Yu a ,1,Qing Zhou a ,b ,1,Yimei Cai a ,b ,Qibin Luo a ,c ,Hongbin Lin a ,Songnian Hu a ,?,Jun Yu a ,?

a Key Laboratory of Genome Information and Sciences,Beijing Institute of Genomics,Chinese Academy of Sciences,Beijing,100029,China

b Graduated School of the Chinese Academy of Sciences,Beijing,100039,China

Department of Genome Oriented Bioinformatics,Technische Universit?t München,Wissenschaftszentrum Weihenstephan,85350Freising,Germany

a b s t r a c t

a r t i c l e i n f o Article history:

Received 27March 2009Accepted 11August 2009

Available online 19August 2009Keywords:Silkworm microRNAs Origin Cluster Targets

MicroRNAs (miRNAs)are pivotal regulators involved in various physiological and pathological processes via their post-transcriptional regulation of gene expressions.We sequenced 14libraries of small RNAs constructed from samples spanning the life cycle of silkworms,and discovered 50novel miRNAs previously not known in animals and veri ?ed 43of them using stem-loop RT-PCR.Our genome-wide analyses of 27species-speci ?c miRNAs suggest they arise from transposable elements,protein-coding genes duplication/transposition and random foldback sequences;which is consistent with the idea that novel animal miRNAs may evolve from incomplete self-complementary transcripts and become ?xed in the process of co-adaptation with their https://www.sodocs.net/doc/c43940919.html,putational prediction suggests that the silkworm-speci ?c miRNAs may have a preference of regulating genes that are related to life-cycle-associated traits,and these genes can serve as potential targets for subsequent studies of the modulating networks in the development of Bombyx mori .

Introduction

MicroRNAs (miRNAs)are a class of endogenous short non-coding transcripts that contain intramolecular complementary foldbacks.Mature miRNAs,processed by Drosha and Dicer,are recruited to the RNA-induced silencing complex (RISC)and guides RISC to the target genes [1,2].In Drosophila melanogaster and Caenorhabditis elegans ,a subset of miRNAs are produced from mirtrons,which are short debranched introns that mimic the pre-miRNAs and bypass canonical Drosha processing [3,4].Most pre-miRNAs are derived from either introns or intergenic regions;some miRNA genes may form clusters and are transcribed as a single polycistronic transcript [5].

miRNAs have been fascinate objects for research ever since their discovery as they have powerful functions in physiological and pathological processes via post-transcriptional regulation of gene expression [6–8].Different from their plant counterparts that bind to target genes with nearly perfect complement [9,10],animal miRNAs identify targets via ～7-nt complements to their 5′ends.Although the functional mechanism of miRNAs has not been fully understood yet,some experiment evidence revealed that the functions and mechan-isms of animal miRNA-mediated gene regulation include blockage of translational initiation,translational repression after initiation,co-translational degradation,target deadenylation coupled with tran-script degradation,and sequestration from ribosomes (by relocalizing

into P/GW-bodies)[11].It is widely accepted that different miRNA genes can give birth to similar or even identical mature miRNAs,implying that they may share overlapping functions [12].However,it is unlikely the case because miRNAs are deployed their speci ?cities of precise temporal and spatial expressions during developmental stages as some duplicated protein-encoding genes.For instance,members of the let-7family in nematode all have similar 5′seed sequences and thus have overlapped target genes.They are expressed at different stages during development,and their particular temporal expression pattern underlies their essential roles in stage-speci ?c developmental traits.It has been discovered that three members (miR-48,miR-84and miR-241)of the let-7family are activated between the second (L2)and the third (L3)larval stages of the C .elegans ;they promote L3development by repressing hbl-1.However,let-7is activated between the L4and the adult stages,and it promotes adult development by suppressing lin-41and hbl-1[13,14].Another case is the K box gene family of Drosophila ,which are expressed at spatial levels.In Droso-phila embryonic development,miR-13b-1and miR-13b-2can produce an identical mature miRNA,though,they exhibit non-overlapping expression patterns in central nervous system and muscles,respectively [15,16].Therefore,diverse functions of miRNAs of the same family can be accomplished,at least in part,by recruitment in different temporal and spatial domains.

Several hypotheses have been brought forward to explain the origin of miRNAs.The emergence of certain Arabidopsis thaliana miRNAs relies on the duplication of genic regions or the duplication/transposition of a gene that has been subjected to a previous duplication event [17–19].Recent evidence has shown that new miRNAs of A .thaliana arose from sequences that either have self-

Genomics 94(2009)438–444

?Corresponding authors.Fax:+861082995373.

E-mail addresses:husn@https://www.sodocs.net/doc/c43940919.html, (S.Hu),junyu@https://www.sodocs.net/doc/c43940919.html, (J.Yu).1

Xiaomin Yu and Qing Zhou contributed equally to this

10.1016/j.ygeno.2009.08.007

Contents lists available at ScienceDirect

Genomics

j o u r n a l h o m e p a g e :w ww.e l s e v i e r.c o m /l o c a t e /y g e n o

complementarity by chance or represent highly degenerate inverted duplications[20,21].In animals,miRNAs may originate from random sequences after enough accumulation of mutations[22],or directly from transposons[23–25].This is not at all surprising,as animal genomes are capable of encoding a large amount of candidate hairpins that are plausible for pre-miRNAs[15,26,27].Extensive cloning evidence has revealed that number of incidental hairpins that grow into components of miRNA processing pathways is very limited. Although these hairpins can form miRNAs and then?nd corresponding target genes,their regulatory capacities would be subject to natural selections;only those that provide useful functions to the organism may eventually evolve into genuine miRNA genes [28–30].

Although a large fraction of miRNAs are evolutionarily conserved, direct cloning of small RNAs also revealed that many miRNAs in plants and animals are species-speci?c[19,31,32].Here,we present the discovery in silkworm(Bombyx mori)and other insects of50novel miRNAs by small RNA cloning of tissue samples collected at different life stages of the organism.Among the50miRNAs,27are unique to silkworm(i.e.,not found in?ies,bee,beetle,mosquito,worms or?sh). Potential functions of these27Bm-speci?c miRNAs were analyzed by target gene predictions.Our results suggest that some miRNAs are development-related regulators and species-speci?c miRNAs may play important roles in particular life history traits of the silkworm. Based on origin analysis,we propose that young Bm-speci?c miRNAs stem from duplicated protein-coding genes,transposable elements, and random foldback sequences.

Results and discussion

Novel silkworm miRNAs and a spectacular miRNA cluster To explore novel miRNAs in the silkworm genome,we re-mapped a total of2877short sequences that previously cannot be annotated by mapping to miRBase12.0and silkworm genome V1.0and V2.0.Three conserved miRNAs(bmo-miR-9c,bmo-miR-306and bmo-miR-989) and50novel miRNA candidates were identi?ed throughout the14 developmental stages(Table1).These novel miRNAs have not been previously discovered in animals and most of them were cloned in a restricted stage except bmo-miR-2733-1which was detected in both the pre-diapaused egg stage(PDS)and the diapause-broken egg stage (DBS).In addition,most of these novel miRNAs originate from intergenic regions except bmo-miR-2728,bmo-miR-2731a/b,and bmo-miR-2739 which are parts of intron sequences(Table1).All the50miRNA precursors can fold into canonical hairpin structures according to our criteria(Table S1).Lengths of the50miRNAs vary from17-nt to29-nt, with the major part being within18-24-nt(～67.31%)(Fig.1A).At the5′end,there is a preference of the composition of miRNA sequences for a U instead of a G at the?rst position(Fig.1A and B).All these features of the miRNA sequences are in good concord with known speci?city of the Dicer processing[33].Further,given its high sensitivity,stem-loop RT PCR was employed to con?rm the expression of novel miRNAs.Forty-three of the novel miRNAs and two miRNA?yielded PCR products.This approved that they are undoubtedly expressed at corresponding developmental stages(Fig.2).

As we were preparing this manuscript,Zhang et al[34]have published a study on silkworm miRNA and reported an identi?cation of354putative silkworm miRNAs including most conserved miRNAs using a deep sequencing approach with a RNA mixture from egg, larval,pupal,and adult stages.We compared our50novel miRNAs with theirs and found only eight miRNAs(including four mature miRNAs and four pre-miRNAs)were nearly identical(Table S2).We further found that bmo-miR-C-16and bmo-miR-C-25were detected by the two different sequencing methods albeit lacking products from our stem-loop RT-PCR test.We are able to draw two conclusions from these studies:one that emphasizes the difference between the discovery methods and one that suggests that the expressions of many miRNAs are spatiotemporal controlled worthy of more research efforts.

The distribution of miRNAs in the silkworm genome shows that10 miRNA genes(bmo-mir-1920/C-23/C-19/1922/C-7a/b/C-10/C-11a/ b/c/C-20/C-25/C-21)including6silkworm-speci?c ones(bmo-mir-C-23/C-19/1922/C-7a/b/C-10/C-11a/b/c)cluster together in an 8000-bp region of Scaffold1143(Table1and Fig.3).The positional clustering of miRNAs is a common genomic feature of miRNAs[1],and each cluster usually contains two or three miRNAs like the six pairs of silkworm conserved miRNAs clusters(bmo-miR-1/133,bmo-miR-2/ 13,bmo-let-7/100,bmo-miR-12/283,bmo-miR-275/305and bmo-miR-9b/79)previously discovered in silkworm[26],while the similarly large miRNAs clusters also reported in C.elegans[35],D. melanogaster[36]and human[37].The close proximity of these10 novel miRNA genes implies that they may be transcribed and processed as a large precursor under a single regulatory element [38].Such an operon-like organization converges on the same target gene or targets a different number of genes for decreased transcrip-tion and/or translational repression[35].

Bm-speci?c miRNAs and their origin

To inspect the conservation in related species,we used these50 novel miRNAs and seven other silkworm miRNAs(bmo-miR-1920–bmo-miR-1926)previously discovered[26]as seed sequences to search their homologs in the genomes of other insects,worm and?sh.

A total of30miRNAs were found to be conserved among these animals(Table S3);some appear as highly conserved miRNAs,such as bmo-miR-1924that has homologs in eight Drosophila species,and they may share analogous functions in developmental events in these metazoans.

The remaining27silkworm miRNAs are not conserved,and these species-speci?c miRNAs are most likely to have emerged recently.To track the origin of these young miRNA genes,we mapped their precursors to a mixed set of unigenes and transposons as well as to the reference genome.These species-speci?c miRNAs were further divided into two groups according to their similarities to some other genomic/transcriptomic regions(E values≤0.05)(Fig.4).

The?rst group is composed of miRNAs that have high sequencial similarities not only to one or more genomic loci,but also to unigenes and/or transposons sequences.Precursors of seven miRNAs(bmo-mir-1921/C-10/C-11a/c/C-12/C-23/C-5)have at least one arm that has similarity or is complementary to transposons.It is likely, therefore,that these young silkworm miRNA genes may derive from transposable elements or other genomic repeats.Alignments of another nine pre-miRNAs(bmo-mir-C-19/2735/2738/2725/2741/ 2743/2744/2726/2727)show that they are similar or complemen-tary to one or more protein-coding genes,and thus these miRNAs may arise through genes duplication/transposition,which is similar to the case of plant miRNAs[18,19].The remaining four miRNAs in this group(bmo-mir-2724/2747/2748/1923)have arms similar or com-plementary to both unigenes and transposons,and they may be results of incorporations of certain transposable elements into transcripts,although for these miRNAs there exists a trend of accumulation in regions of low gene density[39].

The second group of miRNAs contains miRNAs that do not have signi?cant alignments with the sequences of unigenes or transposons, as they all have one or multiple similar genomic loci where they may be originated directly from sequences that fortuitously harbor certain features of pre-miRNA.

The origin analysis of miRNAs has implications for new miRNA precursors arisen over evolutionary time scale.Similar to plants, some young silkworm miRNAs may evolve from transposable elements or duplicated protein-coding genes as certain small RNAs of C.elegans are proposed to be produced from near-perfect inverted

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repeats[31].In fact,previously computational folding has shown that the silkworm genome could harbor several millions of pre-miRNA-like hairpins[26].Our observations support the idea that animal genomes may comprise scores of evolutionarily transient miRNAs, which frequently arise from transposons,protein-coding gene duplication/transposition or random foldbacks[40].Compared to plants,it may be easier for young miRNAs of animals to derive from arbitrary sequences to regulate their putative target genes after accumulation of positive mutations,because the base-pairing requirement of animal miRNAs with their relevant target genes is much lower than that in plants[20].Potential functions of Bm-speci?c miRNAs

We searched for target genes of the27Bm-speci?c miRNAs in 2598annotated3′UTRs using the PITA program with theΔΔG criterion set to–10kcal/mol and other parameters left to default. Prediction results show that a total of2352genes belonging to1175 gene families were potential targets of the27Bm-speci?c miRNAs. We then selected the target genes expressed at different develop-mental stages and performed another round of prediction.As listed in Table S4,the potential functions of these predicted targets include hormone-biosynthesis and regulation pathways(such as juvenile

Table1

Novel miRNAs expressed in B.mori.

Name Sequence(5′to3′)Development

stages b RT-PCR

identi?cation

Location of pre-miRNA sequences c Intergenic/intron

bmo-miR-2723CCAGCAGUGGACGUCUGUG BKS Yes nscaf2842(+):2054080–2054163(12)Intergenic

bmo-miR-2724UCCCCCGGUGUGAUAGCUCC FLS No nscaf2971(?):232715–232803(8)Intergenic

bmo-miR-2725UGGAAUGAGGUUGCUGCGUCGACAAUU BKS Yes nscaf3063(+):3340673–3340755(16)Intergenic

bmo-miR-2726UGAUUUACAGUUAUGGGACCUUUUAUU PDS No nscaf3097(+):2898562–2898640(28)Intergenic

bmo-miR-2727UAACGAUGUCCUGGAUACGUUUAAUGCUC MLS Yes nscaf3099(?):161224–161314(28)Intergenic

bmo-miR-2753UCAAAUGGAAGAAGCUGGUA BKS Yes nscaf3078(?):312923–313027(4)Intergenic

bmo-miR-C-7a CUAGCAUGUGUGCGAGUCA DBS Yes scaffold1143(+):3527–3611(?)Intergenic

bmo-miR-C-7b AGUCUAGCAUGUGUGCGAGUCA DBS Yes scaffold1143(+):3527–3611(?)Intergenic

bmo-miR-2728UUCAAUUGGAUAGUUGGUGGCUUCAUC NLS Yes nscaf2983(+):2720311–2720400(7)Intron:Bmb023437 bmo-miR-2729AUUUUAUGAGGUCGGUCCAU BS Yes nscaf2901(+):824617–824715(18)Intergenic

bmo-miR-C-10GAAGGCGAACGCUCGACG DS Yes scaffold1143(+):3651–3719(?)Intergenic

bmo-miR-C-11a GGUGAGGGAUGUACGUGGA FLS Yes scaffold1143(+):4589–4659(?)Intergenic

bmo-miR-C-11b AGGUGAGGGAUGUACGUGGA DBS Yes scaffold1143(+):4589–4659(?)Intergenic

bmo-miR-C-11c UUCAGGUGAGGGAUGUA DBS Yes scaffold1143(+):4589–4659(?)Intergenic

bmo-miR-C-12GGUGUCCGGAUACUCUCUGC DBS Yes nscaf2930(+):3727575–3727655(3)Intergenic

bmo-miR-2730GUGAGGUACACACCUCGG TAS Yes scaffold016956(+):5811–5913d(?)Intergenic

bmo-miR-2731a UGCGAGACCAUGGUAUGUGGA NLS Yes nscaf2952(?):70974–71064(?)Intron:Bmb038902 bmo-miR-2731b AACAGAUGCGAGACCAUGGUAUGUGGA BS Yes nscaf2952(?):72108–72198(?)Intron:Bmb009093 bmo-miR-2732UAGCUAAUUGUUGUGGCUAGUCCGGAUG BS Yes nscaf2888(?):8699878–8699962(15)Intergenic

bmo-miR-C-16CGGACGGUAUCAUUAAA DBS No scaffold1143(+):3297–3360(?)Intergenic

bmo-miR-2733-1UCACUGGGUGCAUGAUGAUUGU DBS,PDS Yes scaffold006264(+):15518–15601d(?)Intergenic

bmo-miR-2734UUUGUCGUGUCUGGUAGUCGC DS Yes nscaf2953(?):43885–43973(14)Intergenic

bmo-miR-C-19GGCGUACGUUUACGUGC DBS Yes scaffold1143(+):3110–3208(?)Intergenic

bmo-miR-C-19?GUCGGCACGGUACGUGAA FLS Yes scaffold1143(+):3110–3208(?)Intergenic

bmo-miR-C-20AGCGUUCGAGUUCCAUGGAAU MLS Yes scaffold1143(+):5600–5668(?)Intergenic

bmo-miR-C-21a UUACUAGUCGCGUUUCGCA MLS Yes scaffold1143(+):7308–7373(?)Intergenic

bmo-miR-C-21b CUUUACUAGUCGCGUUUCGCAU PS Yes scaffold1143(+):7308–7373(?)Intergenic

bmo-miR-2735UUGGGAACCAGCUUGGA MLS Yes nscaf3027(?):678228-678322(23)Intergenic

bmo-miR-C-23CAGCACUGAAUCCCGUU MLS Yes scaffold1143(+):2727–2796(?)Intergenic

bmo-miR-C-23?CGGGAGAUGUGGUGUUCGGGAG TAS Yes scaffold1143(+):2727–2796(?)Intergenic

bmo-miR-2736UACCAAAACGUCGAUGGUACCAGCAGU PDS Yes scaffold017403(?):3988–4071d(?)Intergenic

bmo-miR-C-25CUGCUCAGUACGAGAGGAACCGCAG DBS No scaffold1143(+):7076–7157(?)Intergenic

bmo-miR-2737UUUUAUACUGUCCAGAUUUGU SS Yes nscaf3068(?):424548–424630(1)Intergenic

bmo-miR-2738AUCUUUUAGAACGGCCAUCUGAUG MLS Yes nscaf2770(?):900800–900909(?)Intergenic

bmo-miR-2739GAGAUGUGGAUAUUAUGGUGUGGAGG MS Yes nscaf3027(+):1195025–1195122(23)Intron:Bmb012620 bmo-miR-2740CUCAGUUAUGAAACUGCCAUGAC PDS No scaffold18821(?):439–522(?)Intergenic

bmo-miR-2733-2UCACUGGGUGCGUGAUGAUUGU PDS Yes nscaf2176(?):345409–345488(11)Intergenic

bmo-miR-2741UGCAUUGGUGCGGAAUGCCAUCCAUGG BKS Yes nscaf2674(?):287427–287529(5)Intergenic

bmo-miR-2742UUUUCAUUGGAUUAGUGUU BKS Yes nscaf2899(?):169828-169894(?)Intergenic

bmo-miR-9c a UAAAGUUAUGGUACCGAAGUUA SS Yes nscaf2839(+):28891–28998(12)Intergenic

bmo-miR-2743CAUGAGCGAGGUUGGAAAA MLS No Scaffold5448(+):635-778(?)Intergenic

bmo-miR-2744UUUGAGAGUCCUAGCUAG PDS Yes Scaffold27571(+):33–141(?)Intergenic

bmo-miR-2745UAAAUUCGGUCUUUCGGG MLS Yes nscaf2210(?):2415636–2415714(1)Intergenic

bmo-miR-2746UACCGUGAUCAAUUAGGCGG BKS Yes nscaf2800(?):1908910–1909009(27)Intergenic

bmo-miR-2747UGUCAUCGAUGCAUCCAGGGUAACGCCC DBS Yes Scaffold18309(?):93–197(?)Intergenic

bmo-miR-2748UCCGGCGAGAAAACUCAGUGGG PS Yes nscaf2855(?):5705147–5705240(10)Intergenic

bmo-miR-C-40AACGGCGGGAGUAACUAUGACUCU DS Yes scaffold4034(?):480-578(?)Intergenic

bmo-miR-2749CCACGAUCACUCCGACACCAUCCCUGGA PDS Yes scaffold3946(?):443–561(?)Intergenic

bmo-miR-C-42AGAUUCCCACUGUCCCUAU DBS Yes nscaf2176(+):4305438–4305527(11)Intergenic

bmo-miR-306a UCAGGUACUAGGUGACUCUGA LFLS Yes nscaf2839(+):30888–30995(12)Intergenic

bmo-miR-2750AUACAGGCCUUCAAUCUGC BKS No nscaf3035(+):1352662–1352745(24)Intergenic

bmo-miR-C-45CUUGGGAGAAUCAGCGGGGAAA MLS Yes nscaf3098(+):993621–993680(28)Intergenic

bmo-miR-2751GUCUGGGCCGUGGAGCGUUU MLS Yes nscaf2943(?):2570504–2570607(14)Intergenic

bmo-miR-2752CUCAACACGAUGGCCACGGA PDS Yes scaffold13931(+):104–242(?)Intergenic

bmo-miR-989a GUGUGAUGUGACGUAGUGGAA PPS,MS No scaffold4254(+):425–515(13)Intergenic

a bmo-miR-9c,bmo-miR-306and bmo-miR-989are conserved miRNAs identi?ed by homology search.

b Pre-diapaused egg(PDS);diapaused egg(DS);diapause-broken egg(DBS);blastokinesis stage egg(BKS);trachea appearing stage egg(TAS);bluish egg(BS);newly hatched larva(NLS);fourth-instar larva(FLS);molting larva(MLS);late?fth-instar larva(LFLS);spinning larva(SS);pre-pupa(PPS);pupa(PS);moth(MS).

c This row liste

d scaffold ID,strand,start position,end position and chromosom

e ID consecutively.

d Location of pre-miRNAs from genom

e sequences o

f SilkDB_V1.0.

440X.Yu et al./Genomics94(2009)438–444

hormone esterase,farnesyl pyrophosphate syntase,farnesyl diphos-phate synthase,allatostatin preprohormone,ecdysone 20-hydroxy-lase,methyltransferase,cytochrome P450,myosuppressin receptor and RXR type hormone receptor),physiological process (such as vesicle amine transport protein,trehalase,laccase 2,larval cuticle protein,chitinase,apterous ,silk proteinase inhibitor,apoptosis response protein),and signal transduction (such as presenilin enhancer,Wnt -1,notch-like protein,ras -related protein).

In contrast with the conserved miRNAs,these Bm-speci ?c miRNAs may have a function preference for regulating the target genes related

to life-cycle-associated traits.Since the ecdysone and juvenile hormone orchestrates the ecdysis and metamorphosis in the life cycle of silkworm [41],Bm-speci ?c miRNAs,especially those expressed in fourth-instar larva (FLS),molting larva (MLS)and pupa (PS)stages,may bear the function of ?ne-tuning a series of genes that control the levels of ecdysone and juvenile hormone or larval cuticle protein and chitinase to regulate the complicated and transient process of periodic ecdysis and metamorphosis.For example,bmo-miR-C-11a was detected in FLS which comes before MLS,and its potential targets —chitinase precursor,ecdysone 20-hydroxylase and cuticle protein —may be suppressed by bmo-miR-C-11a simultaneously by this miRNA,which results in the forthcoming digestion of unsclerotized layers of the cuticular exoskeleton through the stimulation of chitinase by 20-hydroxyecdysone [42].Such predictions of target genes have shed light on the potential functions of these Bm-speci ?c miRNAs;though,further investigations are needed to expand our understanding of the modulating mechanisms of these miRNAs in the development of silkworm.Materials and methods

Experimental materials of silkworms

The inbred silkworm strain Dazao was provided by the Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhen-jiang,P.R.China.All samples were collected at proper developmental stages as described previously [26].All materials of silkworm used in this study including six embryonic developmental stages (pre-diapause stage-PDS,diapause stage-DS,diapause-broken stage-DBS,blastokinesis stage-BKS,trachea-appearing stage-TAS and bluish stage-BS),?ve larval stages (newly-hatched larva stage-NLS,fourth-instar larva stage-FLS,molting larva stage-MLS,late ?fth-instar larva stage-LFLS and spinning stage-SS),two metamorphic stages (pre-pupa stage-PPS and pupa stage-PS)and one imaginal stage (moth stage-MS).

Cloning of small RNAs and sequence analysis

Cloning of small RNAs was performed as described previously [26].In brief,small RNAs under 40-nt were size-fractionated and enriched with ?ashPAGE ?Fractionator (Ambion,Austin,TX),and then sequentially ligated to 5′and 3′RNA/DNA chimeric oligonucleotide adapters.Reverse transcription was performed after ligation with the adapters,followed by PCR ampli ?cation.PCR products were cloned and transformed into competent cells.Plasmids were extracted from individual colonies and then sequenced.

After data analysis under our initial criteria published previously [26],we collected a total of 2877short sequences including 1719(46%of 3721)sequences that have at least one match and 1158sequences failed to be mapped on SilkDB V1.0(https://www.sodocs.net/doc/c43940919.html,/silkworm/).In this study,we ?rstly compared these unclassi ?ed sequences to miRBase12.0(https://www.sodocs.net/doc/c43940919.html,/)to identify conserved miRNAs,and then remapped them to the silkworm genome in SilkDB V1.0and SilkDB V2.0(https://www.sodocs.net/doc/c43940919.html,/silkdb/).Candidates with perfect matches against these datasets expanded upstream and downstream by 100-nt and then fed to RNAfold [43]to predict their secondary structures with adjusting the sliding window size from 60-nt to 150-nt and comparing the folding energy of each sequence within the window with each other.Finally,sequences with minimum folding energy (mfe)no greater than –20kcal/mol were identi ?ed as candidate miRNAs after careful manual inspection.Novel miRNAs con ?rmation using stem-loop reverse-transcription PCR

cDNA was synthesized from total RNA using miRNA-speci ?c stem-loop primers obtained from commercial service (Takara,Dalian).All

Fig.1.The length distribution and nucleotide compositions of the novel miRNAs.(A)Length distribution of novel miRNAs.Each 5′end nucleotide A,U,G,and C are represented by blue,brown,green,and purple,respectively.(B)Sequence composition logo of the newly identi ?ed miRNAs.The height of each letter is proportional to the frequency of the indicated

nucleotide.

Fig.2.Stem-loop RT PCR con ?rmation of novel miRNAs identi ?ed based on cloning from speci ?c development stages.U6snRNA serves as a positive control.

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Fig.3.The large miRNA cluster in B.mori .Hairpin structures are illustrated with mature miRNAs (highlight in red)corresponding to red solid arrowheads,which are positioned on scaffold

1143.

Fig.4.Detection of Bm-speci ?c miRNA related sequences in the B.mori genome (red),transcriptome (blue)and transposons (green).Each pre-miRNA of silkworm-speci ?c miRNA was aligned using BLAST,and all hits (E values ≤0.05)are reported.Group I contains miRNAs which with highly sequence similarity not only to one or more genomic loci,but also to transcriptome and/or transposons sequences.Group II miRNAs are those that have signi ?cant similarity only to genomic sequence.

442X.Yu et al./Genomics 94(2009)438–444

the stem-loop RT primers and gene-speci?c primers are listed in Table S5.Reverse transcriptase reactions contained20ng of RNA samples, 50nM stem-loop RT primer,1×RT buffer,0.25mM each of dNTPs (Promega),0.01M DTT(Invitrogen),5U/μl SuperScript?II reverse transcriptase(Invitrogen)and0.25U/μl RNase Inhibitor(Promega). The15-μl reactions were incubated in an Applied Biosystems2720 Thermal Cycler in a96-well plate at16°C for30min,42°C for30min, 85°C for5min and then4°C for subsequent process.The cDNAs were 1:15diluted to perform PCR of con?rmation and additional real-time PCR of expression analysis.PCR mixture contains1μl cDNA,0.5μM forward and reverse primers,1×PCR buffer,1.75mM Mg2+,0.25mM each of dNTPs(Promega)and1.25U Taq polymerase(Fermentas). The20μl PCR reactions were performed using Applied Biosystems 2720Thermal Cycler in200μl micro-tubes at95°C for5min,followed by35cycles of15s at95°C,30s at58°C to62°C,and30s at72°C.PCR products were detected by electrophoresis with3%agarose gel containing ethidium bromide and photographed under UV light. Analysis of conserved miRNAs and Bm-speci?c miRNAs

To identify conserved miRNAs in other species,we compared novel silkworm candidate miRNAs to genome sequences of other species including12Drosophila species(http://?https://www.sodocs.net/doc/c43940919.html,),Anopheles gam-biae(https://www.sodocs.net/doc/c43940919.html,/index.php),Apis mellifera (https://www.sodocs.net/doc/c43940919.html,/projects/honeybee),Tribolium cas-taneum(https://www.sodocs.net/doc/c43940919.html,/projects/tribolium),C.ele-gans(https://www.sodocs.net/doc/c43940919.html,)and Danio rerio(http://www. https://www.sodocs.net/doc/c43940919.html,/Projects/D_rerio).We extracted candidate sequences with no more than four internal nucleotides mismatches expanded them to upstream and downstream by100-nt and then predicted their hairpin structures with the same criteria used for the identi?cation of novel silkworm candidate miRNAs.

We test the sequences similarity of the pre-miRNA with the transcriptome,transposons,and reference genome from the silkworm data to track the origin of the Bm-speci?c miRNAs.The matched data are classi?ed into groups.The?rst group contains pre-miRNAs that have≥95%identity to genome sequence and their precursors whose 3′arm and/or5′arm have signi?cant similarity with unigenes and/or transposons(E values≤0.05).The remaining Bm-speci?c pre-miRNAs are categorized into the second group.

Target gene prediction for species-speci?c miRNAs

We extracted3′UTR sequences of silkworm(Dazao strain) UniGene from NCBI UniGene database(http://www.ncbi.nlm.nih. gov/sites/entrez?db=unigene),and the PITA program[44]was employed to predict miRNA targets with parameters set to default. Target genes withΔΔG no greater than–10kcal/mol were selected from the original predictions and then analyzed. Acknowledgments

We would like to thank Professor Anying Xu of the Sericultural Research Institute,Chinese Academy of Agricultural Sciences,for providing silkworm eggs.This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (08SQN01185)awarded to Xiaomin Yu,and a grant from the Ministry of Science and Technology(2006CB910400)awarded to Jun Yu. Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at doi:10.1016/j.ygeno.2009.08.007.References

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TEMSDiscovery2.5操作指南概论

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在导入.cel(小区数据) 文件时的选项:要定义小区数据是属于哪一个project(define target project),然后Browse小区数据。导入过程中，我们会在TASK WINDOW中看到相应的project/.cel导入信息。导入好小区数据之后我们会在project Explorer中看到我们新建的project (20100801)中会出现Composite（组合）/datasets（数据组）,现在这里还是空的,然后我们右键project(比如:20100801)—view/edit properties会看到我们cell configuration已经存在CELL文件了。 ,

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台湾陆续出现电影但放映的都是外国人的影片。直到年北京丰泰照相馆的老板任庆泰以著名京剧艺术家谭鑫培作为拍摄对象拍下了他表演定军山》的几个片断观众反响热烈。这预示了中国纪录片的萌芽。而国际上公认的第一部纪录电影是罗伯特?弗拉哈迪在年拍摄的北方的纳努克》这也是他的第一部电影。直到今天这部电影仍然充满着无穷的魔力被热爱纪录片的专家学者作为研究欣赏图本。原因就在于他开创了纪录片的拍摄手法。纪录片依托电影发展壮大在电影和电视界闯下了一番天地被全世界人民所认同。从年电视发明以后人们就可以足不出户了解世界新闻、博览社会百态。影视合流成了趋势。电视纪录片应电视技术的成熟、媒体力量的聚合诞生了。美国国家地理频道、探索频道依托纪录片而崛起、发展英国的日本的在世界上在纪录片的专属领域中享有美誉。中国的电视杨紫烨现在是中国纪录片最好时代新浪网? 引言纪录片发从年起步至今已走过了五十三年的历史现状又是如何呢研究的目的和意义选择中国电视纪录片的现状作为论文的研究对象其目的和意义在于第一新千年已进入第十一个年头科学技术日新月异中国电视纪录片自身在承载内容和外在形式上都表现出个性化、丰富化的特点通过回顾半个世纪的电视纪录史分析出每个时期电视纪录片的共性站在历史的肩头才能更好的审视现在展望未来。第二电视生态环境与纪录片发展息息相关在市场经济时代纪录片面临着哪些问题又该如何把握自己的话语权这些问题的探讨是纪录片现状生存必须面对的课题。第三电视纪录片是一个复杂动态的

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Discovery纽约时代广场探索博物馆EB-5项目项目概况探索频道（Discovery Channel）于1985年在美国创立，探索频道目前覆盖全球超过160个国家、4亿5千万个家庭，探索公司同时也是美国的上市公司，是美国最大的主流媒体之一。 Discovery博物馆（mDiscovery Times Square）成立于2009年，是探索频道（Discovery）的官方合作伙伴，为纽约市的前五大的博物馆。地处于时代广场核心的44街与第七第八大道中间，过去成功展出：泰坦尼克号、哈利波特、法老王和兵马俑等世界知名展览，已接待超过数百万人次的游客。继成功推出纽约时代广场第一期娱乐项目“百老汇4D剧院”项目（进展顺利，投资者均取得I-526移民申请通过）后，曼哈顿区域中心（MRC）又重磅推出位于纽约时代广场的第二期娱乐项目Discovery博物馆——探索纽约项目，该项目与第一期4D剧院项目仅隔一街距离。

项目特点独一无二的地理优势纽约时代广场在2013年迎接了5340万次游客，游客总消费超过了400亿美金，旅游消费预计将会在未来4年每年以8.5%的速度增长。良好的发展前景——纽约市的旅游统计表

足够的就业机会创造依照Michael Evans所做出的就业人数计算（即RIMS Ⅱ计算方式，该计算方法为美国移民局比较推荐的就业机会计算方式），该项目预计产生593个新的就业机会。远远超过EB-5所需的240个就业机会空间高达60%。银行专户还款 Discovery博物馆参观门票预计价格为22美元，娱乐产业一直以来都是现金流十分可观的产业，依照与其他时代广场相似项目比较并且保守评估推算，每年项目净利润预计高达一千万美元以上，项目承诺在营运方面将保留60%的现金存放至还款账户中，专款专户作为未来贷款五年还款准备。资金结构

DAVID使用方法介绍

DAVID使用说明文档一、DAVID简介 DA VID （the Database for Annotation，Visualization and Integrated Discovery）的网址是https://www.sodocs.net/doc/c43940919.html,/。DA VID是一个生物信息数据库，整合了生物学数据和分析工具，为大规模的基因或蛋白列表（成百上千个基因ID或者蛋白ID列表）提供系统综合的生物功能注释信息，帮助用户从中提取生物学信息。 DA VID这个工具在2003年发布，目前版本是v6.7。和其他类似的分析工具，如GoMiner，GOstat等一样，都是将输入列表中的基因关联到生物学注释上，进而从统计的层面，在数千个关联的注释中，找出最显著富集的生物学注释。最主要是功能注释和信息链接。二、分析工具： DAVID需要用户提供感兴趣的基因列表，在基因背景下，使用提供的分析工具，提取该列表中含有的生物信息。这里说的基因列表和背景文件的选取对结果至关重要。 1.基因列表：这个基因列表可能是上游的生物信息分析产生的基因ID列表。对于富集分析而言，一般情况下，大量的基因组成的列表有更高的统计意义，对富集程度高的特殊Terms有更高的敏感度。富集分析产生的p-value在相同或者数量相同的基因列表中具有可比性。 DAVID对于基因列表的格式要求为每行一个基因ID或者是基因ID用逗号分隔开。基因列表的质量会直接影响到分析结果。这里定性给出好的基因列表应该具有的特点，一个好的基因列表至少要满足以下的大部分的要求：（1）包含与研究目的相关的大部分重要的基因（如标识基因）。

教你DIY中文增强版Geexbox

教你DIY中文增强版Geexbox Geexbox是一款可以从光盘上直接启动的Linux多媒体操作系统【当然也可以从硬盘和USB闪存上启动】，它是基于Linux和MPlayer进行开发应用的，它可以让你不用进windows 就可以欣赏大片。它几乎支持大部分主流媒体格式，包括AVI、RM、RMVB、MPEG-4、MP3及外挂中文字幕，可以让旧电脑变成强悍的媒体中心。可惜官方提供的只有英文的ISO 镜像，因此网上也出现了不少网友定制的中文版。他们是怎么做的呢？其实很简单。利用官方提供的GeeXboX ISO Generator，你也可以轻松DIY属于自己的Geexbox中文增强版。还犹豫什么呢？下面就和笔者一起来体会DIY的乐趣。一、GeeXboX ISO Generator初上手 “工欲善其事，必先利其器”，首先，请你到Geexbox的官方网站（https://www.sodocs.net/doc/c43940919.html,/en/downloads.html）下载最新的GeeXboX ISO Generator。然后将下载到的geexbox-generator-1.0.i386.tar.gz用Winrar解压到硬盘中（本文以“D：\geexbox”为例进行说明）。进入解压目录，双击generator.exe运行软件(这个镜像生成器还包括在Linux和Macosx下使用的程序)。进入程序界面，你可以看到八个标签页，它们分别是：界面设置(Interface)、音频设置（Audio)、视频设置(Video)、遥控设置（Remote control）、网络设置（Network）、服务设置（Services）、液晶显示设置（Lcd display）、套件设置（Packages）。接下来，请你单击“Packages“，进入套件设置项。这里列出的都是一些非常有用却没有包含在压缩包中的解码器（Codecs）、固件（Filmwares）、字体（Fonts）和主题（Themes）。建议你选中所有的解码器、固件、主题以及字体——“Chinese Simplified-GB2312”，然后点击”DownlOad”按钮下载。好啦，沏杯热茶慢慢等，Generator自己会通过网络把相应的文件下载到本地硬盘中。（如图1）心急的朋友如果受不了牛速，你也可以直接进入官方ftp下载所需资源： ⑴.解码器：https://www.sodocs.net/doc/c43940919.html,/codecs/ 将下得的压缩包解压至 D：\geexbox\iso\GEEXBOX\codec\即可。 ⑵.固件：https://www.sodocs.net/doc/c43940919.html,/firmwares/ 将下得的压缩包解压至 D：\geexbox\iso\GEEXBOX\firmwares\即可。 ⑶.字体：https://www.sodocs.net/doc/c43940919.html,/fonts/ 将下得的压缩包解压至 D：\geexbox\i18n\fonts\即可。

纪录片制作机构

探索频道（Discovery Channel）是由探索传播公司（Discovery Communications, Inc./DCI；NASDAQ：DISCA，旗下拥有197多家全球性电视网，包括Discovery探索频道、动物星球频道、Discovery科学频道和Discovery高清视界频道等）于1985年创立的，总部位于美国马里兰州蒙哥马利县银泉市。探索频道主要播放流行科学、科技、历史、考古及自然纪录片。探索频道自1985年在美国启播后、现今已成为世界上发展最迅速的有线电视网络之一、覆盖面遍及全国百分之九十九的有线电视订户、在全球145个国家和地区有超过14400万个家庭订户。探索频道是全球最大的纪录片制作及买家、它吸引全球最优秀的纪录片制作人、所以探索频道的节目均被认为是世界上最优秀的纪实娱乐节目。也是世界上发行最广的电视品牌，目前到达全球160多个国家和地区的30600多万家庭，以35种不同语言播出节目。探索频道在世界主要国家地区均有落地，但探索频道会因应不同地区设立不同版本，加上字幕或配音。美国版本主要播放写实电视节目，如著名的流言终结者系列。亚洲探索频道除着重播放写实节目之外，也播放文化节目，如介绍中国、日本文化的一系列节目。亚洲探索频道于1994年成立，总部在新加坡，为美国Discovery传播公司（DCI）的全资附属机构，提供二十四小时精彩的纪实娱乐节目。据2005年泛亚媒体调查（PAX）的结果显示，探索频道在富裕成人中连续9年被公认为亚洲地区收视人口最多的有线及卫星电视频道。在新加坡举办的2004年“亚洲电视大奖”评选中，探索频道还荣膺“年度最佳有线及卫星电视频道”。中国国际电视总公司（中央电视台全额投资的大型国有独资公司，成立于1984年，是中国内地规模最大、赢利能力最强的传媒公司）境外卫星代理部接收探索频道信号，通过亚太6号卫星（东经134度）发射KU波段信号。该服务一般只提供给三星级或以上的涉外宾馆酒店，外国人居住区，领事馆及大使馆。中国大陆各省市的地方电视台会转播或播放探索频道制作的节目。同时，还与浙江华数集团成立合资公司，向由杭州电视台开办的四个面向全国播出的高清付费电视频道（求索纪录、求索生活、求索科学、求索动物）提供绝大多数的节目内容。

discover微波操作手册

微波合成仪标准操作手册一、操作流程 1、例行检查：仪器开机前，首先检查仪器整体是否正常；反应腔及内衬溢出杯是否清洁；检查自动压控装置APD是否清洁；自动进样器是否在正常位置；仪器电源线、数据线、气体管路连接情况是否正常。经检查一切正常方可开机。如内衬、APD不清洁或其它问题未经处理而运行仪器所造成的损害，属于非正常操作范畴。 2、开机顺序：先打开计算机电源，再打开Discover主机电源，然后运行Synergy软件（在计算机桌面上）。最后打开空压机电源。 3、登记制度：检查、开机均正常，请认真按规定填写仪器使用记录，记录信息不全将承担后续使用问题的责任。检查、开机、运行过程中，发现任何问题请及时联系管理员。 4、启动软件：运行Synergy软件，选择用户名并输入密码，进入软件操作界面后，可从屏幕右下方工具栏察看Discover和Explorer的联机情况。 5、放入样品：按要求装配好微波反应管（详见第六部分），放入仪器衰减器。 6、选择方法：打开软件界面中相应用户的“M ethod”文件夹图标，选择所需方法，单击鼠标左键拖拽到相应样品位置，如有需要，可新建方法或对方法进行修改（详见第四部分） 7、运行前检查：检查衰减器是否处于锁定状态；察看屏幕右侧温度、压力的显示是否正常。 8、运行方法：点击软件界面上部工具栏中的“P lay”按钮，仪器自动运行。二、禁止的操作项 1、严禁频繁开关机；开机后1min内关机；关机后1min内开机。 2、严禁修改电脑系统设置如注册表项等内容。 3、严禁使用破损的、有裂痕的、划痕严重的反应瓶。 4、严禁使用变形的样品盖。 5、反应瓶盖必须严格按要求装配，禁止未经过检查就放置于自动进样器架上。 6、严禁将标签纸粘贴在反应瓶的任何部位。 7、严禁将文献中多模微波仪器（特别是家用微波炉）的反应条件直接用于该仪器。 8、严禁长时间无人值守，仪器运行过程中，必须每2小时进行巡视查看，并做好检查记录。 9、微波程序运行过程中，严禁非仪器管理员在线修改反应参数。 10、仪器登陆用户只有管理员的权限可以设置为“Admin”其他均设置为“User”。 11、仪器各登陆用户的参数设置应符合仪器要求（详见第三部分），禁止修改。

SuperScan 使用教程

扫描工具SuperScan使用教程（如何使用SuperScan） SuperScan 是由Foundstone开发的一款免费的，但功能十分强大的工具，与许多同类工具比较，它既是一款黑客工具，又是一款网络安全工具。一名黑客可以利用它的拒绝服务攻击（DoS，denial of service）来收集远程网络主机信息。而做为安全工具，SuperScan能够帮助你发现你网络中的弱点。下面我将为你介绍从哪里得到这款软件并告诉你如何使用它。如何获得SuperScan SuperScan4.0是免费的，并且你可以在如下地址下载： https://www.sodocs.net/doc/c43940919.html,:81/up/soft3_2010/SuperScan.rar 因为SuperScan有可能引起网络包溢出，所以Foundstone站点声明某些杀毒软件可能识别SuperScan是一款拒绝服务攻击（Dos）的代理。 SuperScan4.0只能在Windows XP或者Windows 2000上运行。对一些老版本的操作系统，你必须下载SuperScan3.0版。 SuperScan的使用给SuperScan解压后，双击SuperScan4.exe，开始使用。打开主界面，默认为扫描（Scan）菜单，允许你输入一个或多个主机名或IP范围。你也可以选文件下的输入地址列表。输入主机名或IP范围后开始扫描，点Play button，SuperScan开始扫描地址，如下图A。

图A：SuperScan允许你输入要扫描的IP范围。扫描进程结束后，SuperScan将提供一个主机列表，关于每台扫描过的主机被发现的开放端口信息。SuperScan还有选择以HTML格式显示信息的功能。如图B。

美国探索教育视频资源服务平台

1、美国探索教育视频资源服务平台平台内容及意义大众文化的流行，娱乐学习一体化的浪潮席卷全球。同时随着社会发展，多学科交叉融合，使得社会对大学生综合能力要求颇高。在某一个方面出类拔萃的复合型人才，越来越受到企业社会的青睐。综合性人才在当今社会炙手可热，因此学校在重视专业课的同时，加强对课外知识的普及符合当今教育时代的发展需求。美国探索教育视频资源服务平台坚持以“科教兴国”为总方略，以提高在校师生综合素质、开拓师生眼界为宗旨；以教育、科学、文化、历史、探险等为题材的多学科交叉融合的教育视频资源服务平台。平台始终坚持科学研究与教学理论相统一，历史知识和文化教育相结合，以求达到师生即使足不出户，亦能知大千世界之神奇、能知世界各地前沿性科学技术，能解世间万物之疑惑。此平台已经成为西安数图网络科技有限公司一个独具特色的教育资源服务平台。平台特色美国探索教育视频资源服务平台，结合高校科学教育及科普知识所需，精选整合美国探索频道（Discovery）和美国国家地理频道（National Geography）两大世界知名频道近年来的最新节目，精心制作而成。 1、美国探索频道（Discovery） 1985年开播使用客户在全球达到160多个国家，3亿零6百多万家庭。通过15颗卫星用36种语言、24小时播放来源于全球不同地方摄制的精彩高品质纪实节目 2、美国国家地理频道（National Geography）遍布全球达171个国家及地区通过48种语言收看荣获1次奥斯卡金像奖和2次金像奖提名，129座艾美奖平台分类自然科学，历史人文，科学发现，生命科学，旅游风光，体育探索，军事侦探，交通机械，工程建筑

discovery教程

第一章：前言 (1) 第二章：微机油藏描述系统集成 (3) 一、Landmark公司微机油藏描述系统发展历程 (3) 二、微机油藏描述系统各模块集成 (4) （一）工区、数据管理系统（二）GESXplorer地质分析与制图系统（三）SeisVision 2D/3D二维三维地震解释系统（四）PRIZM 测井多井解释系统（五）ZoneManager层管理与预测（六）GMAPlus正演建模三、Discovery微机油藏描述系统软件特色 (12) 第三章：微机三维地震解释系统软件应用方案研究 (13) 一、工区建立 (13) （一）工区目录建立（二）一般工区建立（三）工区管理二、数据输入 (20) （一）地质数据输入 1 井头数据输入 2 井斜数据输入 3 分层数据输入 4 试油数据输入 5 生产数据加载 6 速度数据输入（二）测井数据输入 1 ASCII格式测井数据输入 2 LAS格式测井数据输入（三）地震数据输入 1 SEG-Y三维地震数据输入 2 层位数据输入 3 断层数据输入

三、微机地质应用 (31) （一）微机地质应用工作流程工作流程 1 地质分析工作流程 2 沉积相分析工作流程（二）微机地质应用 1 井位图建立 2 等值线图(isomap)建立 3 各种剖面图(Xsection)建立 4 生产现状图制作 5 沉积相图制作四、微机三维地震解释综合应用 (48) （一）微机三维地震解释工作流程 1 合成记录及层位工作流程 2 地震解释工作流程 3 速度分析工作流程（二）微机三维地震解释综合应用 1 地震迭后处理－相干体 2 合成记录制作及层位标定 3 层位和断层建立、解释 4 三维可视化 5 速度分析与时深转换 6 构造成图 7 地震测网图建立 8 地震属性提取五、微机单井测井解释及多井评价 (104) （一）微机单井测井解释及多井评价工作流程 1 测井曲线环境校正与标准化工作流程 2 测井分析流程（二）微机单井测井解释及多井评价 1 打开测井曲线 2 测井曲线显示模板制作 3.测井曲线显示、编辑与预处理 4.交会图制作与分析 5 测井解释模型建立与解释 6 测井解释成果报告

BBC一百多部记录片

BBC一百多部记录片 BBC.生物记录片.细胞 https://www.sodocs.net/doc/c43940919.html,/cszGSiqUkU9cr（访问密码：e215）自然风光喜马拉雅山脉 https://www.sodocs.net/doc/c43940919.html,/cs4iYcAeiHKIn 提取码：28c1自然风光巴厘岛 https://www.sodocs.net/doc/c43940919.html,/csizn3trNnCGv 提取码：e5edBBC纪录片《野性水域终极挑战》[MKV] https://www.sodocs.net/doc/c43940919.html,/Qi24t6zR3TyCK （提取码：bbcb）[历史地理] 詹姆斯·卡梅隆的深海挑战. https://www.sodocs.net/doc/c43940919.html,/lk/cJxR8pIvfSvR8 访问密码4076远方的家-边疆行全100集 https://www.sodocs.net/doc/c43940919.html,/cszGATNBFhjjw（访问密码：52c6）美丽中国湿地行50集

https://www.sodocs.net/doc/c43940919.html,/cszX2JZKa6UVy 访问密码2f2f李小龙：勇士的旅程》(Bruce Lee A Warriors Journey) https://www.sodocs.net/doc/c43940919.html,/csFPTqFZr8GTz 提取码6c71CHC高清纪录片：星球奥秘之地球雪球期MKV 720P 1.4G 英语中字 https://www.sodocs.net/doc/c43940919.html,/QGEpqiPbfGfsG （访问密码：cdb2）探索频道：狂野亚洲：四季森林 https://www.sodocs.net/doc/c43940919.html,/cJxPJZXa8wGzA 访问密码1034BBC 纪录片《美国的未来》[MKV/4集全] https://www.sodocs.net/doc/c43940919.html,/QivxnUNbqLEau （提取码：27fe）生命的奇迹.全5集 https://www.sodocs.net/doc/c43940919.html,/cJXTIkq5jLBY5 访问密码7d2f《华尔街》高清收藏版[HDTV/720p/MKV/全10集] https://www.sodocs.net/doc/c43940919.html,/cy5PrZeud43Rk 提取码8497远方的家-沿海行(高清全112集) https://www.sodocs.net/doc/c43940919.html,/cszX4jUKD29ay 访问密码a52aBBC

全球最好的电视台

全球著名电视台掌门人：霍珂灵标签：文化国家电视台（TV station /television station ）指的是制作电视节目并通过电视或网络播放的媒体机构。它由国家或商业机构创办的媒体运作组织，传播视频和音频同步的资讯信息，这些资讯信息可通过有线或无线方式为公众提供付费或免费的视频节目。其播出时间固定，节目内容一部分为其自己制作，也有相当部分为外购。比较有名的电视台：CNN，BBC，TVB，CCTV等。美国有线电视新闻网(ＣＮＮ ) ＣＮＮ由特德·特纳于１９８０年创办，１９９５年被时代—华纳公司兼并。总部设在美国佐治亚州首府亚特兰大市，在美国本土以外设有２８个分部，在世界各地的雇员达４０００人。ＣＮＮ使用英语和西班牙语广播，它的资金来源于用户付费和广告收入。ＣＮＮ因独家报道１９９１年海湾战争而成为家喻户晓的有线新闻广播公司，目前已覆盖全球２１０个国家和地区。 ? 什么叫CNN? ?CNN是什么？ ?CNN什么意思啊好像最近很流行还有什么流行词啊？ ?美国的CNN公司是什么东西请消息说明一下 ?CNN 是美国的还是法国的 ?CNN歪曲报道原文英国广播公司(BBC) 这一新闻频道由英国广播公司于１９９１年成立。它在海外拥有２５０名记者和５８个分部，资金来源于用户付费和广告收入。该频道声称在全球拥有２．７亿个家庭用户。英国广播公司今年宣布，计划于２００７年新开播一个阿拉伯语的新闻频道。 ? BBC是什么？ ?BBC什么意思 ?BBC是什么啊 ?BBC是哪个国家的媒体哦? ?bbc的经典语录(games[TV]的BBC) ?求bbc所有纪录片目录半岛电视台(ＡｌＪａｚｅｅｒａ) 半岛电视台由卡塔尔政府于１９９６年成立。它在全球雇有１７０名记者，拥有２６个分部。世界各地都能收看到半岛电视台的阿拉伯语频道。半岛电视台因不断报道伊拉克和中东其他地区的一些事件而遭到美国的指责。美国总统布什甚至曾计划轰炸它的卡塔尔总部。２００６年，该电视台还将推出英语频道。 ?半岛电视台的相关资料？ ?卡塔尔半岛电视台与cctv ?为什么半岛电视台收视率全球第一？cctv1呢? ?基地组织为什么要把拉登的录音送到半岛电视台? ?半岛电视台在中东哪里？据说很有名的！ ?半岛电视台是哪国的欧洲新闻电视台(Ｅｕｒｏｎｅｗｓ) 欧洲新闻电视台建立于１９９３年，它的特点之一就是使用英语、法语、德语、意大利语、葡萄牙语、西班牙语和俄语７种语言播报新闻。该电视台所以能这样做是因为它主要使用各个通讯社提供的图像，而没有亮相屏幕的新闻主播。该电视台由１９个欧洲公共部门电视频道共同所有，总部设在法国城市里昂，雇

纪录片是否要完全真实

纪录片不一定要完全真实对于纪录片真实性的鉴定，就犹如不同的人看《哈姆雷特》，每个人都有自己的看法，而我的观点是：纪录片不一定要完全真实。我在这里提到的完全真实是指没有摆拍，没有编排。我认为纪录片中可以存在重现，摆拍。有种对纪录片的定义是：一切真实记录社会和自然事物的非虚构的电影片或电视片都是纪录片。对于非虚构的电影片或电视片就可能存在编排和摆拍。我的想法在国外和少数中国导演那里可以得到些许的认可。在国外，纪录片是很受欢迎的，甚至纪录片的频道需要付费。就拿众所周知的美国的Discovery探索频道为例，美国的Discovery探索频道于1985年开播，是世界上发行最广的电视品牌，目前到达全球160多个国家和地区的3亿零6百多万家庭，以35种不同语言播出节目。美国的Discovery探索频道的很多纪录片就是摆拍，重现的。Discovery有一档栏目叫重案夜现场，这个栏目并不是完全跟拍警方的破案过程，而是进行情景再现的，以摆拍，采访的方式进行重述。在这个节目里事件是真实的，专家的口述是真实的，而犯罪现场的以及犯罪证据，甚至犯罪过程的还原都是情景再现的，除了重案夜现场，历史零时差，与恐龙共舞特别篇等等都是情景再现的方式。情景再现即编排和摆拍。

黑格尔曾经说过：真实不是别的，而是缓慢的成熟过程。我觉得这句话，对于中国的纪录片仍然是很实用的。在我们国家，为什么人们不喜欢看纪录片？我想很大原因是因为我们国家的纪录片很多是不成熟的，但是有些导演的纪录片是很招人喜欢的，比如张以庆导演的影片《英和白》《幼儿园》《周周的世界》，冷冶夫的《伴》《油菜花开》等等，那么他们的影片是否是完全真实的呢？冷冶夫在接受采访时说，他的《油菜花开》：“基本全部是摆拍，因为它是一种实验纪录片，国外翻译过来是“真实电影”，这种纪录片除了载体好以外，它的故事也好。我在主流媒体做的都是纪实风格的纪录片，很多人看不到我的另一面，所以我今天斗胆地放了这样一部片子”。当记者问到：“那您觉得摆拍还叫纪录片吗？”冷冶夫答道：“其实国际上往往把有没有这件事作为纪录片的鉴定。写剧本拍摄，那属于虚构的故事片，如果有这么件事，不管你怎么弄，它都是属于非虚构类的。国外对纪录片的分类特别粗，你也可以看到，包括国外那些Discovery节目几乎都用了情景再现的方式。” 我个人喜欢看《油菜花开》这样的纪录片，首先它的镜头很美，假如是跟拍，想必一定没有这么美的镜头；其次选材更容易，事件的结局知道，就更容易分析这件事件，就更容易找到切入点，在接下来编排摆拍时就更容易制造氛围，从而达到教育感化等效果，如果从开始就跟拍的纪录片，不一定能准确料定时间的结局，就不容易分析事件。张以庆导演的纪录片一直以选材新颖，立意深刻著称，他肯花大

优秀自然纪录片

自然纪录片（这里面又大概分为地球、宇宙、人体三个大部分）一、BBC地球篇首先是BBC三大“镇馆之宝”（自封的）《地球脉动》：几乎算是有史以来最好的生态纪录片，用接近上帝的视角，审视这个叫做地球的星球，虽然探讨的是科学，但是有着宗教式的观影体验。《人类星球》：一部极其特别的自然纪录片，说是讲地球，其实是在从社会学的角度讲人类，但说是讲人类的纪录片，它又是以自然生态地理环境等要素为载体来讲述。视点新颖，内容丰富，把自然和人文结合得天衣无缝。《生命》：人类看完足以无地自容的片子，哪怕只是地穴里一只微不足道的小虫子，每天也在上演生存的史诗。为了吃饭，为了繁衍，为了活下去，无数精彩甚至悲壮的生物行为，在这个生生不息的地球上无限地演绎下去，地球也因此而不朽。除了我心目中难以超越的三大神作之外，仍有一些不能不看的作品。《植物之歌》：这部讲植物的算是侧重于动物的《生命》的姊妹篇，从植物的进化讲到对地球，对生态的影响，这是一次对地球的绿色，也是对生命的礼赞。《非洲》：我认为最接近三大神作的作品，拍得极其出色。非洲大地上生命的瑰丽，壮阔，惊奇，灵动，不朽，一一呈现在镜头前。此外，大量蒙太奇，慢镜头的运用，让这部纪录片的观赏性和趣味性，也达到一个难以超越的高度。（例如，那个从沙丘底部拼命往上推粪球的屎壳郎君，一次次往上推，粪球一次次滚落，好不容易推上沙丘，一阵风吹过又把它直接吹到底部。表现得非常有趣，但笑着笑着不知不觉眼泪就出来了。。。）《冰冻星球》：算是《地球脉动》和《生命》的一条支线，讲述两极大陆的世界与“居民”们。极地世界的镜头难能可贵，摄制组捕捉到了很多平常难以观测到的动物捕食、迁徙等活动，以及壮观的冰川与雪原，还有对全球变暖趋势的忧虑。《蓝色星球》：这部纪录片的人气在国内不如以上几部那么高，但是其品质足以名列BBC前茅。本片深入海洋，对水下蔚蓝的世界进行深入细致的介绍。从起源到各方各面，再到反思，每一集主题鲜明，节奏得当，配乐非常动听，本片足以成为自然纪录片的教科书。 ==========题外话的分割线===========================

Discovery软件介绍与基本操作

Discovery软件介绍与基本操作一、软件的整体综合评价Discovery油藏描述软件具有工区管理、数据加载、地质分析、地震综合解释和测井分析的工作思路和操作流程，作为一体化油藏描述系统，每一部分工作都不是孤立地进行的，而是彼此之间数据、成果必须相互调用、相互参考。Discovery软件功能强大，具有以下几方面的优点： 1、模块较多而且集成化，有地震、地质、测井和油藏多方面的集成环境与综合交叉应用。 2、数据库系统的一体化，各类数据统一管理，调用。 3、成果共享，各个功能模块之间的解释处理成果可以相互导入，实现无逢衔接，协同工作。尽管有很多优点，但也存在一些缺陷和瑕疵：1、软件的设计风格和设计思路不太符合我们的应用习惯，很难短期快速接受。2、有些功能还不太完善，对于某些复杂的操作和综合智能控制，还稍显得粗糙。3、菜单操作烦琐，较少的快捷键，而且还不能自定义。二、软件的安装Discovery软件可以在微机上应用，对硬件的要求也不太高，方便灵活。在安装的过程中，要特别注意选择安装模式和米制单位。完成之后，要记得把Excel加载宏gxdb.xla文件拷贝到office文件夹的系统库里面，为以后Excel表格数据的输入做准备。三、工区的建立和管理这个步骤是工作的基础，非常重要，关键是不要对工区的四角范围限制，设置为“not set”，方便扩展，要选择正确的辽河油区的坐标系统。刚建立的新工区是自动激活的，当我们需要在其他工区工作时，首先关掉正在运行的已激活工区下的各个模块窗口，然后再去activate目标工区，防止机器报错。工区备份打包和恢复非常实用，易于数据的保存和移植。四、各模块的论述与应用 1．数据加载（1）地质数据的加载wellbaseWellbase能加多种数据，常用的是井头、井斜、分层和生产试油数据。Excel表格输入关键是把握数据类型，数据的起始行，对wellid的命名不能用中文、不能重复，加了之后不能修改。井斜数据在加入后，需要calculate偏移量，才能看到斜井的井轨迹。分层采用的一般是顶分层，与我们习惯应用的不一样，对井缺失的层位，可以没有数据。（2）测井数据的加载prizm测井数据可以加载系统已经有的曲线，也能加预先定义的任何曲线。常用的是ASCII码和las格式，las格式可以批量输入，自动完成。ASCII码要指定wellid，与井匹配，而且还要定义曲线名称和单位，给出空值和采样间隔，对于不需要的列可以skip。（3）地震数据的加载seisvision首先要把SEG-Y数据转换成3dx或3dh格式，在这个过程中，给出数据类型，是时间域还是频率域的。输入线号和道号，通过head dump数据体能知道这些信息。 2．Prizm技术应用（1）曲线显示调整我们可以把曲线的排列布局，也就是曲线的标题栏按需拖拽到理想的位置，放到合适的Track里面。通过查看曲线的直方图，得到最大值和最小值，据此来调整上下限的值，使曲线全部显示出来。另外可以调网格的样式，曲线的vertical 显示比例，曲线道的宽度，曲线形式和颜色。增加或删除Log或Linear性质的Track，设置左右包络线和充填pattern，进行区域充填。通过录井网下载一个90格式的岩性代码文件，然后把它转成las格式导入，再对曲线充填就能得到一个岩性柱子。最后把设置好的显示结果另存为一个template，并且在setting菜单里设为project的默认模板，以后打开其他的井，就不用再调了。（2）曲线校正编辑曲线的重采样，只要设置新的采样间隔interval和步长step，就能显示并

discovery中文教程

4.找到实例数据文件后，单击Open.。将出现General Properties（一般属性）对话框用于输入ASCII文本文件的定义。 5.在General Properties对话框中输入以下信息: z数据格式的Name（名）称为New Wells（新井）。 z在Description（描述）框中输入以下信息: 文件包括Well ID、 Operator（作业者）、Well Name（井名）、Well Number（井号）和 Status （状态）等一般井信息。每个字段用逗号分开。文本文件井头在第一行。

z Application是一种Well数据格式。这项选择决定在把ASCII数据映射到数据库中（或LandNet的层）时你的有效目标是什么。接受这个默认值。 z正常情况下，这些ASCII文本文件带有一个.txt后缀。在Default Extension(s) for ASCII Data Files（ASCII数据文件默认扩展名中）可输入txt, .txt, or *.txt. 输入该信息后，输入数据时选择ASCII数据文件会默认地查找*.txt文件。 z DefCon2 正确判断记录分隔符的末端和记录类型。一个ASCII数据文件中记录的基本格式。但Record ID选择的默认值需要改为(none), 因为数据文件的所有记录的格式相同。 z将Number of Header Records（井头记录序号）改为1用来说明包含列标题的ASCII 文本文件的第一行。 z正确的字段分隔符为逗号字段，正确的字段限定符为none（无）。完成后的General Properties（一般属性）对话框应该像以下对话框。 6.单击OK。将出现Records Definition（记录定义）窗口。ASCII文本文件的每个字段或数据列将在这个窗口中进行定义。

STM32F0-DISCOVERY用户手册

1/30 文档ID 022910第1版2012年3 月 STM32F0DISCOVERY STM32F0探索套件 UM1525 前言 STM32F0DISCOVERY 是意法半导体STM32F0系列微控制器的探索套件，用于帮助你探索STM32F0 Cortex-M0微控制器的功能，轻松开发应用设计。STM32F0探索套件基于1颗STM32F051R8T6微控制器，组件包括ST-LINK/V2嵌入式调试工具、LED 指示灯、按键和1个原型板。图1： STM32F0 探索套件用户手册

2/30UM1525 文档ID 022910第1版目录目录 1. 约定....................................................................................................................................52. 快速入门 (6) 2.1 开始使用........................................................................................................ 62.2 系统要求..........................................................................................................62.3 支持STM32F0探索套件的开发工具链 .......................................................62.4 订货代码. (6) 3. 特性....................................................................................................................................74. 硬件与原理图.. (8) 4.1 STM32F051R8T6 微控制器 ..........................................................................114.2 嵌入式ST-LINK/V2编程器/调试器 . (13) 4.2.1 使用ST-LINK/V2向板载STM32F0烧录和调试代码 ............................14 4.2.2 使用ST-LINK/V2向外部STM32应用板烧录和调试代码. (15) 4.3电源和电源选择............................................ 164.4 LED 指示灯 ...................................................................................................164.5 按键................................................................................................................164.6 JP2（Idd ）﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍﹍ 16 4.7 OSC 时钟 -----------------------------------------------------------------------------174.7.1 OSC 时钟电源 .............................................................................................174.7.2 OSC 32kHz 时钟电源 17 4.8 焊桥.................................................................................................................184.9 扩展连接器.. (19) 5. 尺寸图..............................................................................................................................266. 原理图..............................................................................................................................277. 修改历史记录 (30)