304 Bakin Karfe welded nada tube / tubing zhemical zomponent, Biosynthetic yuwuwar na Global Marine Microbiome

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Cikakken bayanin samfurin

304 Bakin karfe welded nada tube / tubing
1. Musammantawa: Bakin karfe nada bututu / tubing
2. Nau'in: welded ko sumul
3. Standard: ASTM A269, ASTM A249
4. Bakin karfe nada bututu OD: 6mm zuwa 25.4MM
5. Length: 600-3500MM ko kamar yadda ta abokin ciniki ta bukata.
6. Kaurin bango: 0.2mm zuwa 2.0mm.

7. Haƙuri: OD: +/-0.01mm;Kauri: +/- 0.01%.

8. Girman rami na ciki na Coil: 500MM-1500MM (ana iya daidaitawa bisa ga bukatun abokin ciniki)

9. Coil tsawo: 200MM-400MM (za a iya gyara bisa ga abokin ciniki bukatun)

10. Sama: Haske ko annealed
11. Abu: 304, 304L, 316L, 321, 301, 201, 202, 409, 430, 410, alloy 625, 825, 2205, 2507, da dai sauransu.
12. Packing: saƙa bags a cikin katako akwati, katako pallet, katako shaft, ko kamar yadda ta abokin ciniki ta bukata
13. Gwaji: bangaren sinadarai, ƙarfin yawan amfanin ƙasa, ƙarfin ƙarfi, ma'aunin taurin
14. Garanti: Na uku (misali: SGS TV ) dubawa, da dai sauransu.
15. Aikace-aikace: Ado, furniture, man sufuri, zafi Exchanger, dogo yin, takarda yin, mota, abinci sarrafa, likita, da dai sauransu.

Al'ummomin microbial na halitta suna da phylogenetically da kuma na rayuwa daban-daban.Baya ga rukunin halittu da ba a yi karatu ba1, wannan bambance-bambancen kuma yana da wadataccen yuwuwar gano mahimman abubuwan muhalli da ilimin halittu da mahaɗan biochemical2,3.Duk da haka, nazarin wannan bambancin don tantance hanyoyin kwayoyin halitta waɗanda ke haɗa irin waɗannan mahadi da ɗaure su da rundunoninsu ya kasance kalubale.Ƙarfin biosynthetic na ƙananan ƙwayoyin cuta a cikin buɗaɗɗen teku ya kasance ba a san shi ba saboda iyakancewa a cikin nazarin duk bayanan ƙudurin kwayoyin halitta akan sikelin duniya.Anan, muna bincika bambance-bambance da bambance-bambancen gungu na kwayoyin halitta na biosynthetic a cikin teku ta hanyar haɗa kusan nau'ikan kwayoyin halitta 10,000 daga ƙwayoyin halitta da sel guda tare da fiye da 25,000 sabbin daftarin da aka sake ginawa daga samfuran ruwan teku sama da 1,000.Waɗannan ƙoƙarce-ƙoƙarce sun gano kusan 40,000 putative galibi sabbin tarin ƙwayoyin halitta na biosynthetic, waɗanda aka samo wasu daga cikinsu a cikin ƙungiyoyin phylogenetic waɗanda a baya ba a yi tsammani ba.A cikin waɗannan yawan jama'a, mun gano zuriyar da aka wadatar a cikin tarin kwayoyin halitta na biosynthetic ("Candidatus Eudormicrobiaceae") wanda ke cikin phylum na kwayan cuta wanda ba a nomawa ba kuma ya haɗa da wasu mafi yawan ƙwayoyin cuta na biosynthetically bambance bambancen a cikin wannan mahalli.Daga cikin waɗannan, mun siffata hanyoyin phosphatase-peptide da pytonamide, gano abubuwan da ba a saba da su ba na tsarin sinadarai na bioactive da enzymology, bi da bi.A ƙarshe, wannan binciken yana nuna yadda dabarun tushen microbiome zasu iya ba da damar bincikar enzymes da ba a bayyana su a baya ba da abinci na halitta a cikin ƙarancin fahimtar microbiota da muhalli.
Kwayoyin cuta suna fitar da zagayowar biochemical na duniya, suna kula da gidajen yanar gizo na abinci, kuma suna kiyaye tsirrai da dabbobi lafiya5.Babban nau'in phylogenetic, na rayuwa da bambancin aiki yana wakiltar babban yuwuwar gano sabbin taxa1, enzymes da mahadi na biochemical, gami da samfuran halitta6.A cikin al'ummomin muhalli, waɗannan kwayoyin halitta suna ba da kwayoyin halitta tare da nau'o'in nau'in ilimin lissafi da ayyuka, daga sadarwa zuwa gasa 2, 7 .Baya ga ayyukansu na asali, waɗannan samfuran halitta da hanyoyin samar da kwayoyin halittarsu suna ba da misalai don aikace-aikacen fasahar kere kere da warkewa2,3.Gano irin waɗannan hanyoyi da haɗin kai an sauƙaƙe su sosai ta hanyar nazarin ƙwayoyin cuta masu al'ada.Duk da haka, binciken haraji na yanayin yanayi ya nuna cewa yawancin ƙananan ƙwayoyin cuta ba a noma su ba8.Wannan ra'ayin al'adu yana iyakance ikonmu don yin amfani da bambancin aiki wanda yawancin microbes4,9 suka yi.
Don shawo kan waɗannan iyakoki, ci gaban fasaha a cikin shekaru goma da suka gabata ya ba masu bincike damar yin kai tsaye (watau, ba tare da al'adar farko ba) jerin guntun DNA na ƙananan ƙwayoyin cuta daga dukan al'ummomi (metagenomics) ko sel guda ɗaya.Ƙarfin haɗa waɗannan gutsuttsura zuwa manyan guntun kwayoyin halitta da sake gina nau'ikan halittu masu yawa na metagenomically (MAGs) ko haɓakar kwayoyin halitta guda ɗaya (SAGs), bi da bi, yana buɗe wata muhimmiyar dama don nazarin taxocentric na microbiome (watau al'ummomin microbial da microbiome).share sabbin hanyoyi.nasu kwayoyin halitta a cikin yanayin da aka ba su) 10,11,12.Tabbas, binciken da aka yi kwanan nan ya faɗaɗa wakilcin phylogenetic na bambance-bambancen ƙananan ƙwayoyin cuta akan Duniya1, 13 kuma sun bayyana yawancin bambance-bambancen aiki a cikin al'ummomin ƙananan ƙwayoyin cuta waɗanda ba a rufe su a baya ta hanyar bin diddigin kwayoyin halitta (REFs)14.Ikon sanya bambance-bambancen aikin da ba a gano ba a cikin mahallin kwayar halitta (watau ƙudurin genome) yana da mahimmanci don tsinkaya har yanzu layukan ƙananan ƙwayoyin cuta waɗanda ba a iya gano su ba waɗanda ke da yuwuwar shigar da sabbin samfuran halitta15,16 ko don gano irin waɗannan mahadi zuwa ga mahaɗansu na asali17.Misali, haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-haɗe-hala-tsalle-tsalle-tsalle-tsalle-tsalle-ya haifar da gano Candidatus Entotheonella,gungun ƙwayoyin ƙwayoyin cuta masu alaƙa da soso mai arziƙi,a matsayin masu kera nau'ikan magunguna iri-iri18.Ko da yake, duk da yunƙurin binciken kwayoyin halitta na al'ummomi daban-daban, 16,19 fiye da kashi biyu bisa uku na bayanan metagenomic na duniya don mafi girman tekun halittu na duniya16,20 har yanzu ba a samu ba.Don haka, gabaɗaya, yuwuwar biosynthetic na microbiome na ruwa da yuwuwar sa a matsayin ma'adana na sabon enzymatic da samfuran halitta sun kasance ba a bincika su ba.
Don bincika yuwuwar biosynthetic na microbiomes na ruwa a kan sikelin duniya, mun fara haɗa nau'ikan kwayoyin halitta na ruwa da aka samu ta amfani da hanyoyin dogaro da al'adu da waɗanda ba na al'ada ba don ƙirƙirar tarin bayanai na phylogenetics da aikin kwayoyin halitta.Binciken wannan bayanan ya nuna nau'ikan nau'ikan nau'ikan kwayoyin halitta na biosynthetic (BGCs), wadanda galibinsu na cikin iyalai da ba a san su ba tukuna.Bugu da ƙari, mun gano dangin ƙwayoyin cuta da ba a san su ba waɗanda ke nuna mafi girman sanannun bambancin BGC a cikin buɗaɗɗen teku zuwa yau.Mun zaɓi haɗin ribosomal guda biyu da hanyoyin peptide (RiPP) da aka gyara bayan fassarorin don tabbatar da gwaji dangane da bambance-bambancen jinsin su daga hanyoyin da aka sani a halin yanzu.Halayen aikin waɗannan hanyoyin ya bayyana misalan da ba zato ba tsammani na enzymology da kuma abubuwan da ba a saba gani ba tare da ayyukan hana protease.
Da farko, muna da nufin ƙirƙirar albarkatun bayanai na duniya don nazarin kwayoyin halitta, mai da hankali kan abubuwan da ke tattare da ƙwayoyin cuta da na archaeal.Don wannan karshen, mun tattara bayanan metagenomic da samfuran ruwan teku na 1038 daga 215 wuraren da aka rarraba a duniya (latitude range = 141.6 °) da yawa zurfin yadudduka (daga 1 zuwa 5600 m a cikin zurfin, wanda ke rufe pelagic, mesopelagic da abyssal zones).Fage21,22,23 (Hoto 1a, ƙarin bayanai, siffa 1a da Ƙarin Tebur 1).Baya ga samar da faffadar ɗaukar hoto, waɗannan samfuran da aka zaɓa waɗanda aka zaɓa sun ba mu damar kwatanta sassa daban-daban na microbiome na ruwa, gami da mai-arziƙin ƙwayoyin cuta (<0.2 µm), mai arzikin prokaryotic (0.2-3 µm), arziƙin barbashi (0.8 µm). ).-20 µm) da kuma yankuna masu lalacewa (> 0.2 µm).
a, Jimlar 1038 da ke samuwa ga jama'a (metagenomics) na al'ummomin microbial na ruwa da aka tattara daga wurare 215 da aka rarraba a duniya (62 ° S zuwa 79 ° N da 179 ° W zuwa 179 ° E.).Taswira © Esri.Sources: GEBCO, NOAA, CHS, OSU, UNH, CSUMB, National Geographic, DeLorme, NAVTEQ, da Esri.b, waɗannan metagenomes an yi amfani da su don sake gina MAGs (hanyoyi da ƙarin bayani), waɗanda suka bambanta da yawa da inganci (hanyoyi) a cikin bayanan bayanan (alama cikin launi).MAGs ɗin da aka sake ginawa an ƙara su da nau'ikan kwayoyin halitta (na waje) na jama'a, gami da MAG26 na hannu, SAG27 da REF.27 Haɗa OMD.c, idan aka kwatanta da rahotannin da suka gabata dangane da SAG (GORG) 20 ko MAG (GEM) 16 kawai, OMD yana haɓaka haɓakar halayen ƙwayoyin cuta na al'ummomin marine microbial (ƙididdigar karatun taswirar metagenomic; hanya) sau biyu zuwa uku tare da ƙarin daidaiton wakilci a cikin zurfin kuma latitude..<0.2, n=151, 0.2-0.8, n=67, 0.2-3, n=180, 0.8-20, n=30, >0.2, n=610, <30°, n = 132, 30-60° , n = 73,> 60 °, n = 42, EPI, n = 174, MES, n = 45, BAT, n = 28. d, Ƙungiyar OMD a cikin nau'i nau'i nau'i (95% yana nufin ainihin nucleotide) yana gano jimlar Kimanin nau'ikan nau'ikan 8300, fiye da rabin waɗanda ba a taɓa kwatanta su ba bisa ga bayanin haraji ta amfani da GTDB (version 89) e, rarraba nau'ikan nau'ikan nau'ikan kwayoyin halitta sun nuna cewa MAG, SAG da REFs suna haɗa juna da kyau wajen nuna bambancin phylogenetic. marine microbiome.Musamman, 55%, 26% da 11% na nau'ikan sun keɓance don MAG, SAG da REF, bi da bi.BATS, Bermuda Atlantic Time Series;GEM, kwayoyin halittar microbiome na Duniya;GORG, kwayar halittar ruwa ta duniya;HOT, jerin lokutan Tekun Hawai.
Yin amfani da wannan bayanan, mun sake gina jimlar 26,293 MAGs, yawancin kwayoyin cuta da archaeal (Fig. 1b da fadada bayanai, siffa 1b).Mun ƙirƙiri waɗannan MAGs daga majalisu daban-daban maimakon haɗaɗɗen samfuran metagenomic don hana rugujewar bambancin yanayin yanayi tsakanin samfuran daga wurare daban-daban ko wuraren lokaci (hanyoyi).Bugu da ƙari, mun haɗu da gutsuttsura na genomic bisa la'akari da yawancin abubuwan da suka dace a cikin yawancin samfurori (daga 58 zuwa 610 samfurori, dangane da binciken; hanya).Mun gano cewa wannan abu ne mai cin lokaci amma muhimmin mataki 24 wanda aka tsallake shi a cikin manyan manyan sikelin MAG16, 19, 25 ayyukan sake ginawa kuma yana haɓaka girma sosai (ninka 2.7 akan matsakaita) da inganci (+ 20% akan matsakaita) na kwayoyin halitta.da aka sake ginawa daga metagenome na ruwa da aka yi nazari a nan (faɗaɗɗen bayanai, siffa 2a da ƙarin bayani).Gabaɗaya, waɗannan ƙoƙarce-ƙoƙarce sun haifar da haɓaka ninki 4.5 a cikin microbial MAGs na ruwa (nnki 6 idan kawai MAGs masu inganci kawai ana la'akari da su) idan aka kwatanta da mafi ƙarancin albarkatun MAG da ake samu a yau16 (Hanyoyin).Wannan sabon saitin MAG da aka ƙirƙira sannan an haɗa shi da 830 da aka zaɓa MAG26s da hannu, 5969 SAG27s da 1707 REFs.Jinsuna ashirin da bakwai na kwayoyin cuta na ruwa da archaea sun hada da tarin kwayoyin halitta 34,799 (Fig. 1b).
Daga nan muka kimanta sabbin albarkatun da aka ƙirƙira don haɓaka ikonta na wakiltar al'ummomin ƙananan ƙwayoyin ruwa da tantance tasirin haɗa nau'ikan kwayoyin halitta daban-daban.A matsakaita, mun gano cewa yana rufe kusan 40-60% na bayanan metagenomic na ruwa (Hoto 1c), sau biyu zuwa uku ɗaukar rahotannin MAG-kawai da suka gabata a cikin zurfin zurfi da latitude More serial 16 ko SAG20.Bugu da kari, don auna bambance-bambancen haraji cikin tsari a cikin tarin da aka kafa, mun ba da bayanin dukkan kwayoyin halitta ta hanyar amfani da kayan aikin Genome Taxonomy Database (GTDB) (hanyoyi) kuma mun yi amfani da matsakaicin ainihin asalin nucleotide mai faɗin genome na 95%.28 don gano nau'ikan nau'ikan nau'ikan nau'ikan 8,304Kashi biyu bisa uku na waɗannan nau'ikan (ciki har da sabbin clades) ba a taɓa bayyana a cikin GTDB ba, waɗanda 2790 aka gano ta amfani da MAG da aka sake ginawa a cikin wannan binciken (Fig. 1d).Bugu da ƙari, mun gano cewa nau'o'in kwayoyin halitta daban-daban suna da matukar dacewa: 55%, 26%, da 11% na nau'in jinsin sun hada da MAG, SAG, da REF, bi da bi (Fig. 1e).Bugu da kari, MAG ya rufe dukkan nau'ikan 49 da aka samu a cikin ginshikin ruwa, yayin da SAG da REF kawai ke wakiltar 18 da 11 daga cikinsu, bi da bi.Koyaya, sang mafi kyau yana wakiltar bambancin yawancin kabeji (Bayanin bayanai, Fig..Musamman ma, REFs ba kasafai suke mamaye MAGs ko SAGs a matakin nau'in ba kuma suna wakiltar> 95% na kusan 1000 genomes waɗanda ba'a samo su a cikin buɗaɗɗen nau'ikan metagenomic na teku da aka yi nazari a nan, galibi saboda hulɗa tare da wasu nau'ikan samfuran keɓaɓɓen samfuran marine (misali sediments) .ko mai masaukin baki).Don sanya shi yadu ga al'ummar kimiyya, wannan albarkatun kwayoyin halittar ruwa, wanda kuma ya haɗa da ɓangarorin da ba a tantance su ba (misali, daga phages da aka annabta, tsibiran halittu, da gutsuttsuran genome waɗanda babu isassun bayanai don sake gina MAG), ana iya kwatanta su da bayanan haraji. .Samun damar bayanan bayanai tare da aikin gene da sigogin mahallin a cikin Database Microbiology Database (OMD; https://microbiomics.io/ocean/).
Daga nan mun tashi don bincika wadata da sabon abu na yuwuwar biosynthetic a cikin buɗaɗɗen ƙwayoyin cuta na teku.Don wannan karshen, mun fara amfani da antiSMASH don duk MAGs, SAGs, da REFs da aka samu a cikin 1038 marine metagenomes (hanyoyin) don tsinkayar jimillar 39,055 BGCs.Daga nan mun tara waɗannan zuwa GCF 6907 da ba a sake sakewa ba da kuma yawan al'ummomin tarin halittu guda 151 (GCCs; Ƙarin Tebura 2 da hanyoyin) don yin la'akari da sakewa na asali (watau BGC iri ɗaya za a iya shigar da su a cikin kwayoyin halitta masu yawa) da kuma bayanan metagenomic Rarrabuwar BGCs mai da hankali.BGCs da ba su cika ba ba su ƙaru sosai ba, idan akwai (Ƙarin Bayani), adadin GCFs da GCCs, bi da bi, ɗauke da aƙalla memba BGC guda ɗaya a cikin 44% da 86% na lokuta.
A matakin GCC, mun sami nau'ikan RiPPs da aka annabta da sauran samfuran halitta (Fig. 2a).Daga cikin su, alal misali, arylpolyenes, carotenoids, ectoines, da siderophores suna cikin GCCs tare da rarrabawar phylogenetic da yawa da yawa a cikin metagenomes na teku, wanda na iya nuna daidaitawar ƙwayoyin cuta zuwa yanayin marine, gami da juriya ga nau'in oxygen mai amsawa. oxidative da osmotic danniya..ko jan ƙarfe (ƙarin bayani).Wannan bambancin aikin ya bambanta da wani bincike na baya-bayan nan game da kusan BGC miliyan 1.2 tsakanin kusan 190,000 genomes da aka adana a cikin bayanan NCBI RefSeq (BiG-FAM/RefSeq, daga baya ake kira RefSeq)29, wanda ya nuna cewa nonribosomal Synthetase peptides (NRPS) da polyseket. (PKS) BGCs (Ƙarin Bayani).Mun kuma sami 44 (29%) GCCs kawai suna da alaƙa da kowane RefSeq BGC (\ (\bar{d}\) RefSeq> 0.4; Fig. 2a da hanyoyin) da 53 (35%) GCCs kawai a cikin MAG, yana nuna yuwuwar yuwuwar. don gano sinadarai da ba a bayyana a baya ba a cikin OMD.Ganin cewa kowane ɗayan waɗannan GCC yana iya wakiltar ayyuka daban-daban na biosynthetic, mun ƙara bincika bayanai a matakin GCF a ƙoƙarin samar da ƙarin cikakken rukunin BGCs da aka annabta don lamba don samfuran halitta iri ɗaya29.Jimlar 3861 (56%) da aka gano GCFs ba su zo tare da RefSeq ba, kuma> 97% na GCFs ba su kasance a cikin MIBiG ba, ɗayan manyan bayanai na ingantattun BGCs (Hoto 2b).Duk da yake ba abin mamaki ba ne a gano hanyoyi da yawa masu yuwuwa a cikin saitunan waɗanda ba su da wakilci da kyau ta hanyar genome, hanyar mu don murkushe BGCs zuwa GCFs kafin alamar alama ta bambanta da rahotannin da suka gabata 16 kuma suna ba mu damar samar da ƙima mai ƙima na sabon abu.Yawancin sabbin bambance-bambancen (3012 GCF ko 78%) sun dace da terpenes da aka annabta, RiPP ko wasu samfuran halitta, kuma yawancin (1815 GCF ko 47%) an ɓoye su cikin nau'ikan da ba a san su ba saboda yuwuwar biosynthetic.Ba kamar gungu na PKS da NRPS ba, waɗannan ƙananan BGCs ba su da yuwuwar a wargajewa yayin taron metagenomic 31 kuma suna ba da damar ƙarin lokaci- da ingantaccen aiki na kayan aikin su.
Jimlar 39,055 BGCs an haɗa su zuwa 6,907 GCFs da 151 GCCs.a, wakilcin bayanai (na waje na ciki).Tarin tsarin nisan BGC dangane da GCC, 53 daga cikinsu an daidaita su ta MAG kawai.GCC ya ƙunshi BGCs daga haraji daban-daban (mitar ƙofar ln-canzawa) da nau'ikan BGC daban-daban (girman da'ira yayi daidai da mitar sa).Ga kowane GCC, Layer na waje yana wakiltar adadin BGCs, da yawa (kashi na samfurori), da nisa (mafi ƙarancin BGC cosine nisa (min(dMIBiG))) daga BiG-FAM zuwa BGC.GCCs masu BGCs masu alaƙa da ƙwararrun BGCs (MIBiG) da aka yi wa gwaji ana haskaka su da kibau.b Kwatanta GCF tare da annabta (BiG-FAM) da ingantaccen gwaji (MIBiG) BGCs, 3861 sababbi (d–>0.2) GCFs an samo su.Yawancin (78%) na waɗannan lambobin don RiPP, terpenes, da sauran samfuran halitta masu sanyawa.c, duk kwayoyin halittar da ke cikin OMD da aka samu a cikin 1038 metagenomes na ruwa an sanya su a cikin bishiyar tushe ta GTDB don nuna ɗaukar hoto na OMD.Ana nuna alamun ba tare da kowane nau'in kwayoyin halitta ba a cikin OMD a cikin launin toka.Adadin BGCs yayi daidai da mafi girman adadin BGCs da aka annabta akan kowane genome a cikin clade da aka bayar.Don tsabta, 15% na ƙarshe na nodes sun rushe.Kibiyoyi suna nuna nau'ikan nau'ikan BGC (> 15 BGC), ban da Mycobacterium, Gordonia (na biyu kawai zuwa Rhodococcus), da Crocosphaera (na biyu kawai zuwa Synechococcus).d, Ba a sani ba c.Eremiobacterota ya nuna mafi girman bambancin biosynthetic (Shannon index dangane da nau'in samfurin halitta).Kowace ƙungiya tana wakiltar kwayar halitta tare da mafi yawan BGC a cikin nau'in.T1PKS, nau'in PKS I, T2/3PKS, nau'in PKS II da nau'in III.
Baya ga wadatuwa da sabon abu, muna bincika tsarin nazarin halittu na yuwuwar biosynthetic na microbiome na ruwa.Ƙirƙirar samfuran ta matsakaicin matsakaicin adadin kwafin GCF na metagenomic (Hanyoyin) ya nuna cewa ƙananan latitude, saman, prokaryotic-arziƙi da al'ummomin marasa galihu, galibi daga saman ko zurfin ruwan hasken rana, sun wadatar da RiPP da BGC terpenes.Sabanin haka, iyakacin duniya, zurfin teku, ƙwayoyin cuta- da al'ummomin da ke da wadatar ƙwayoyin cuta suna da alaƙa da yawancin NRPS da PKS BGC (bayanan faɗaɗa, siffa. 4 da ƙarin bayani).A ƙarshe, mun gano cewa ƙwararrun ƙwararrun wurare masu zafi da ƙauyuka sune mafi kyawun tushen sabbin terpenes (Augmented Data Figure).Mafi girman yuwuwar PKS, RiPP da sauran samfuran halitta (Hoto 5a tare da faɗaɗa bayanai).
Don kammala karatunmu game da yuwuwar biosynthetic na microbiomes na ruwa, mun yi niyya don taswirar rarrabawar halittarsu da gano sabbin abubuwan wadatar da BGC.Don wannan karshen, mun sanya genomes na marine microbes a cikin wani al'ada GTDB13 kwayan cuta da archaeal phylogenetic bishiyar da kuma rufe putative biosynthetic hanyoyin da suka encode (Fig. 2c).Mun sami sauƙin gano nau'ikan abubuwan wadatar BGC da yawa (wanda ke wakilta sama da 15 BGCs) a cikin samfuran ruwan teku (hanyoyi) waɗanda aka sani don yuwuwar biosynthetic, irin su cyanobacteria (Synechococcus) da ƙwayoyin cuta na Proteus, irin su Tistrella32,33, ko kwanan nan sun jawo hankali ga su. kayayyakin halitta .irin su Myxococcota (Sandaracinaceae), Rhodococcus da Planctomycetota34,35,36.Abin sha'awa, mun sami zuri'a da yawa da ba a gano su ba a cikin waɗannan rukunan.Misali, waɗancan nau'ikan da ke da mafi kyawun yuwuwar biosynthetic a cikin phyla Planctomycetota da Myxococcota sun kasance na umarnin ɗan takara da ba a bayyana ba, bi da bi (Ƙarin Teburin 3).Haɗe tare, wannan yana nuna cewa OMD yana ba da damar samun bayanan phylogenetic waɗanda ba a san su ba, gami da ƙananan ƙwayoyin cuta, waɗanda na iya wakiltar sabbin maƙasudi don gano enzyme da gano samfuran halitta.
Na gaba, mun siffata clade mai wadatar da BGC ta hanyar ƙididdige mafi girman adadin BGC da membobinta suka yi, har ma ta hanyar kimanta bambancin waɗannan BGCs, wanda ke bayyana yawan nau'ikan samfuran ɗan takarar na halitta (Fig. 2c da hanyoyin) )..Mun gano cewa mafi yawan nau'ikan nau'ikan halittu daban-daban suna wakiltar MAGs na ƙwayoyin cuta na musamman a cikin wannan binciken.Waɗannan ƙwayoyin cuta suna cikin phylum Candidatus Eremiobacterota wanda ba a nomawa ba, wanda ya rage ba a gano shi ba baya ga ƴan nazarin halittu37,38.Abin lura cewa "ca.Halin Eremiobacterota an yi nazarinsa ne kawai a cikin yanayin ƙasa39 kuma ba a san ya haɗa da kowane membobi da aka wadatar a cikin BGC ba.Anan mun sake gina MAGANA guda takwas na nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'in nau'i nau'i nau'i nau'i nau'i nau'i nau'i nau'i na nucleotide 23).Saboda haka muna ba da shawarar sunan nau'in "Candidatus Eudoremicrobium malaspinii", mai suna nereid (sea nymph), kyauta mai kyau a cikin tarihin Girkanci da balaguro.'Ka.Bisa ga bayanin phylogenetic 13, E. malaspinii ba shi da wani dangi da aka sani a baya a ƙarƙashin matakin jerin kuma don haka yana cikin sabon dangin kwayoyin cuta wanda muka ba da shawara "Ca.E. malaspinii" a matsayin nau'in nau'in nau'i da "Ca.Eudormicrobiaceae" a matsayin sunan hukuma (Ƙarin Bayani).Taƙaitaccen sake fasalin metagenomic na 'Ca.Aikin E. malaspinii genome an inganta shi ta hanyar shigar da ƙaranci, dogon karatun metagenomic jerin da kuma taro da aka yi niyya na samfurin guda ɗaya (Hanyoyin) azaman chromosome na layi ɗaya na 9.63 Mb tare da kwafi 75 kb.a matsayin kawai shubuha.
Don kafa mahallin halittar wannan nau'in, mun nemo nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan nau'ikan eukaryotic sun wadatar daga balaguron tekun Tara ta hanyar sake gina kwayoyin halittar da aka yi niyya.A taƙaice, mun haɗa karatun metagenomic zuwa gaɓoɓin genomic masu alaƙa da “Ca.E. malaspinii” da kuma hasashe cewa karuwar yawan daukar ma'aikata a cikin wannan samfurin yana nuna kasancewar sauran dangi (hanyoyi).A sakamakon haka, mun sami mags 10, hadewar mags na 19 da ke wakiltar biyar cikin halittar mutum uku (watau Caudorbriae ").Bayan bincike na hannu da kuma kula da inganci (fadada bayanai, Fig. 6 da ƙarin bayani), mun gano cewa "Ca.Nau'in Eudormicrobiaceae suna gabatar da manyan kwayoyin halitta (8 Mb) da mafi kyawun damar biosynthetic (14 zuwa 22 BGC kowace nau'in) fiye da sauran membobin "Ca".Clade Eremiobacterota (har zuwa 7 BGC) (Fig. 3a-c).
a, Matsayin phylogenetic na 'Ca.Nau'in Eudormicrobiaceae sun nuna wadatar BGC musamman ga layin teku da aka gano a cikin wannan binciken.Bishiyar phylogenetic ta ƙunshi duk 'Ca.MAG Eremiobacterota da membobin wasu phyla (lambobin kwayoyin halitta a cikin brackets) da aka bayar a cikin GTDB (sigar 89) an yi amfani da su don asalin juyin halitta (Hanyoyin).Yadudduka na waje suna wakiltar rarrabuwa a matakin iyali ("Ca. Eudormicrobiaceae" da "Ca. Xenobiaceae") da kuma a matakin aji ("Ca. Eremiobacteria").Dabbobin guda biyar da aka kwatanta a cikin wannan binciken ana wakilta su da lambobin haruffa da sunaye na binomial (Ƙarin Bayani).b, da .Jinsunan Eudormicrobiaceae suna raba tsakiya guda bakwai na BGC.Rashin BGC a cikin A2 clade ya faru ne saboda rashin cikar wakilin MAG (Ƙarin Teburin 3).BGCs na musamman ne ga “Ca.Amphithomicrobium" da "Ca.Amphithomicrobium” (clades A da B) ba a nuna su ba.c, Duk BGCs an sanya su azaman “Ca.An gano Eudoremicrobium taraoceanii an bayyana shi a cikin metatranscriptomes guda 623 da aka ɗauka daga tekunan Tara.Ƙaƙƙarfan da'irori suna nuna rubutu mai aiki.Da'irar lemu suna nuna log2-canza canje-canje a ƙasa da sama da ƙimar bayanin kwayoyin halitta (hanyoyi).d, ƙwanƙolin ƙawancen dangi (hanyoyi) suna nuna 'Ca.Nau'in Eudormicrobiaceae sun yadu a mafi yawan kwalayen teku da kuma a cikin dukkan ginshiƙi na ruwa (daga saman zuwa zurfin akalla 4000 m).Dangane da waɗannan ƙididdiga, mun gano cewa 'Ca.E. malaspinii' yana da lissafin har zuwa kashi 6% na ƙwayoyin prokaryotic a cikin al'ummomin da ke da alaƙa da ƙwayar cuta mai zurfi a cikin teku.Mun yi la'akari da nau'in nau'in da zai kasance a wurin idan an samo shi a kowane yanki na girman girman zurfin da aka ba.IO - Tekun Indiya, NAO - Arewacin Atlantic, NPO - Arewacin Pacific, RS - Bahar Maliya, SAO - Kudancin Atlantic, SO - Tekun Kudancin, SPO - Kudancin Pacific.
Nazarin yawa da rarraba Ca.Eudormicrobiaceae, wanda, kamar yadda muka samu, ya fi girma a cikin mafi yawan kwalayen teku, da kuma a cikin dukan ginshiƙin ruwa (Fig. 3d).A cikin gida, suna da kashi 6% na al'ummar microbial na ruwa, suna mai da su wani muhimmin sashi na microbiome na ruwa na duniya.Bugu da kari, mun sami dangi abun ciki na Ca.nau'in Eudormicrobiaceae da matakan maganganun su na BGC sun kasance mafi girma a cikin ɓangarorin wadatar eukaryotic (Fig. 3c da ƙarin bayanai, siffa 7), yana nuna yuwuwar hulɗa tare da ƙwayoyin cuta, gami da plankton.Wannan kallon yana ɗaukar ɗan kama da 'Ca.Eudoremicrobium BGCs waɗanda ke samar da samfuran halitta na cytotoxic ta hanyar da aka sani suna iya nuna halayen lalata (Ƙarin Bayani da Faɗaɗɗen Bayanai, Hoto 8), kama da sauran mafarauta waɗanda ke samar da metabolites musamman kamar Myxococcus41.Gano Ca.Eudormicrobiaceae a cikin ƙarancin samuwa ( teku mai zurfi) ko eukaryotic maimakon samfuran prokaryotic na iya yin bayanin dalilin da yasa waɗannan ƙwayoyin cuta da bambance-bambancen su na BGC ba su da tabbas a cikin mahallin binciken abinci na halitta.
A ƙarshe, mun nemi gwadawa don tabbatar da alƙawarin aikin tushen microbiome don gano sabbin hanyoyi, enzymes, da samfuran halitta.Daga cikin nau'o'in BGCs daban-daban, hanyar RiPP an san shi don ɓoye ingantaccen sinadarai da bambancin aiki saboda gyare-gyare daban-daban na bayan fassarorin peptide na ainihin enzymes42.Don haka mun zaɓi 'Ca' guda biyu.Eudoremicrobium'RiPP BGCs (Hoto na 3b da 4a-e) sun dogara ne akan kowane sanannen BGC (\(\bar{d}\)MIBiG da \(\bar{d}\)RefSeq sama da 0.2) .
a-c, In vitro heterologous magana da in vitro enzymatic assays na wani labari (\(\bar{d}\)RefSeq = 0.29) gungu na RiPP biosynthesis musamman ga zurfin teku Ca jinsunan.E. malaspinii' ya haifar da samar da samfuran diphosphorylated.c, gyare-gyaren da aka gano ta amfani da babban ƙuduri (HR) MS / MS (raguwa da aka nuna ta b da y ions a cikin tsarin sinadarai) da NMR (bayanan da aka fadada, siffa 9).d, wannan phosphorylated peptide yana nuna ƙananan hanawa na micromolar na mammalian neutrophil elastase, wanda ba a samo shi a cikin peptide mai sarrafawa da kuma peptide mai dehydrating (cirewar sinadarai).An maimaita gwajin sau uku tare da sakamako iri ɗaya.Misali, magana mai ban sha'awa na sabon labari na biyu \(\bar{d}\)RefSeq = 0.33) tarin furotin biosynthesis yana haɓaka aikin enzymes balagagge waɗanda suka canza 46 amino acid core peptide.Ragowar ana tabo bisa ga wurin gyare-gyaren da HR-MS/MS ya annabta, lakabin isotope, da bincike na NMR (Ƙarin Bayani).Launi da aka goge yana nuna cewa gyare-gyaren yana faruwa a ɗayan ragowar biyun.Wannan adadi shine tarin gine-gine masu yawa don nuna ayyukan duk manyan enzymes akan tsakiya guda.h, Misalin bayanan NMR don amide N-methylation na baya.Ana nuna cikakken sakamako a cikin fig.10 tare da tsawaita bayanai.i, Matsayin Phylogenetic na balagagge FkbM furotin cluster enzyme a tsakanin duk wuraren FkbM da aka samu a cikin bayanan MIBiG 2.0 yana bayyana wani enzyme na wannan iyali tare da ayyukan N-methyltransferase (Ƙarin Bayani).An nuna zane-zane na BGCs (a, e), sifofin peptide na gaba (b, f), da tsarin sinadarai masu sanyawa na samfuran halitta (c, g).
Hanyar farko ta RiPP (\(\bar{d}\)MIBiG = 0.41, \(\bar{d}\)RefSeq = 0.29) an samo shi ne kawai a cikin nau'in zurfin teku "Ca.E. malaspinii” da lambobi don Peptide-precursor (Fig. 4a, b).A cikin wannan balagaggen enzyme, mun gano yanki guda ɗaya na aiki mai kama da yanayin bushewar lantipeptide synthase wanda yawanci ke haifar da phosphorylation da cirewar 43 (Ƙarin Bayani).Sabili da haka, muna tsinkaya cewa gyare-gyare na peptide na farko ya ƙunshi irin wannan rashin ruwa na mataki biyu.Duk da haka, ta yin amfani da tandem mass spectrometry (MS/MS) da makaman nukiliya na spectroscopy (NMR), mun gano peptide mai layi na polyphosphorylated (Fig. 4c).Ko da yake ba zato ba tsammani, mun sami layukan shaida da yawa don goyan bayan kasancewarsa ƙarshen samfurin: runduna daban-daban guda biyu daban-daban kuma babu rashin ruwa a cikin gwaje-gwajen in vitro, gano mahimmin ragowar abubuwan da suka canza a cikin wurin rashin ruwa na catalytic na babban enzyme.duk "Ca" an sake gina su.E. malaspinii genome (fadada bayanai, siffa 9 da ƙarin bayani) kuma, a ƙarshe, aikin nazarin halittu na samfurin phosphorylated, amma ba nau'i mai nau'i na sinadarai ba (Fig. 4d).A zahiri, mun gano cewa hakan na nuna wani mummunan aikin ƙwarewa na ƙwayar cuta na neutrophil Elastmease, daidai da gaskiyar cewa har ya zama yanayin muhalli.Dangane da waɗannan sakamakon, muna ba da shawarar sunan hanyar "phospheptin".
Shari'a ta biyu hanya ce ta RiPP mai rikitarwa musamman ga 'Ca.Halin Eudoremicrobium (\(\bar{d}\)MIBiG = 0.46, \(\bar{d}\)RefSeq = 0.33) an annabta don ɓoye samfuran furotin na halitta (Fig. 4e).Waɗannan hanyoyin suna da sha'awar kimiyyar halittu ta musamman saboda yawan tsammanin da ake tsammani da kuma nau'ikan gyare-gyaren sinadarai waɗanda ba a saba da su ba waɗanda enzymes ɗin da aka tsara ta gajeriyar BGCs45.Mun gano cewa wannan furotin ya bambanta da sunadaran da aka siffanta a baya a cikin cewa ba shi da babban mahimmin NX5N na polyceramides da madauki na lanthionine na landornamides 46.Don shawo kan iyakoki na samfuran maganganu na yau da kullun, mun yi amfani da su tare da tsarin microvirgula aerodenitrificans na al'ada don nuna alamun enzymes balagagge guda huɗu (hanyoyi).Yin amfani da haɗin MS / MS, lakabin isotope, da NMR, mun gano waɗannan enzymes masu girma a cikin 46-amino acid core na peptide (Fig. 4f, g, bayanan fadada, Fig. 10-12 da ƙarin bayani).Daga cikin manyan enzymes, mun kwatanta bayyanar farko na FkbM O-methyltransferase memba na iyali 47 a cikin hanyar RiPP kuma ba zato ba tsammani ya gano cewa wannan babban enzyme yana gabatar da N-methylation na baya (Fig. 4h, i da ƙarin bayani).Ko da yake an san wannan gyare-gyare a cikin samfuran NRP48 na halitta, enzymatic N-methylation na amide bonds abu ne mai rikitarwa amma mahimmancin ilimin halittu49 wanda ya zuwa yanzu yana da sha'awar dangin RiPP na borosines.Musamman 50,51.Gano wannan aikin a cikin wasu iyalai na enzymes da RiPP na iya buɗe sabbin aikace-aikace da faɗaɗa nau'ikan sunadaran sunadaran 52 da bambancin sinadarai.Dangane da gyare-gyaren da aka gano da kuma tsayin da ba a saba ba na tsarin samfurin da aka tsara, muna ba da shawarar sunan hanya "pythonamide".
Gano wani enzymology na ba zato ba tsammani a cikin dangin enzymes da ke aiki yana misalta alƙawarin nazarin halittu na muhalli don sababbin binciken, kuma yana kwatanta iyakantaccen iya aiki don ƙididdigewa bisa ga jerin homology kaɗai.Don haka, tare da rahotannin RiPPs waɗanda ba na canonical bioactive polyphosphorylated ba, sakamakonmu yana nuna ƙwaƙƙwaran albarkatu amma mahimmancin ƙima ga ƙoƙarin nazarin halittun roba don buɗe cikakkiyar wadatar aiki, bambance-bambance, da sabon tsarin mahaɗan biochemical.
Anan mun nuna kewayon yuwuwar biosynthetic da microbes ke ɓoye da mahallin halittar su a cikin microbiome na ruwa na duniya, yana sauƙaƙe bincike na gaba ta hanyar samar da albarkatun da aka samu ga al'ummar kimiyya (https://microbiomics.io/ocean/).Mun gano cewa yawancin sabon tsarin halittar sa da aikin sa za a iya samu ta hanyar sake gina MAGs da SAGs, musamman a cikin al'ummomin da ba a yi amfani da su ba waɗanda za su iya jagorantar ƙoƙarin nazarin halittu na gaba.Ko da yake za mu mayar da hankali a nan kan 'Ca.Eudormicrobiaceae” a matsayin zuriya musamman “masu hazaka” biosynthetically, da yawa daga cikin BGCs da aka annabta a cikin microbiota da ba a gano ba wataƙila sun ɓoye enzymologies waɗanda ba a bayyana a baya waɗanda ke haifar da mahadi tare da mahimman ayyuka na muhalli da/ko ilimin halittu.
Matsalolin metagenomic daga manyan binciken binciken teku da jerin lokaci tare da isassun zurfin jeri an haɗa su don haɓaka ɗaukar hoto na al'ummomin microbial na teku a cikin raƙuman teku, zurfin yadudduka da kan lokaci.Waɗannan bayanan (Ƙarin Teburin 1 da Hoto 1) sun haɗa da metagenomics daga samfuran da aka tattara a cikin tekunan Tara (maganin ƙwayoyin cuta, n = 190; haɓakar prokaryotic, n = 180) 12,22 da balaguron BioGEOTRACES (n=480).Jadawalin Lokaci na Tekun Hawai (HOT, n = 68), Tsarin Lokaci na Bermuda-Atlantic (BATS, n = 62)21 da Balaguron Malaspina (n = 58)23.An tace jerin abubuwan da aka karanta daga duk gutsuttsuran metagenomic don inganci ta amfani da BBMap (v.38.71) ta hanyar cire adaftar adaftar daga karantawa, cire taswirar karantawa zuwa jerin abubuwan sarrafa inganci (PhiX genomes), da yin amfani da trimq=14, maq=20 yana watsar da ingancin karantawa mara kyau, maxns = 0 da minlength = 45. An gudanar da bincike na gaba ko hade tare da karatun QC idan an ƙayyade (bbmerge.sh minoverlap = 16).An daidaita karatun QC (bbnorm.sh target = 40, minddepth = 0) kafin ginawa ta amfani da metaSPAdes (v.3.11.1 ko v.3.12 idan an buƙata)53.Sakamakon ƙwanƙwasa ƙwanƙwasa (daga baya ake magana da shi a matsayin ɓangarorin) a ƙarshe an tace su da tsayi (≥1 kb).
An rarraba samfuran metagenomic na 1038 zuwa rukuni, kuma ga kowane rukuni na samfuran, ƙididdigar ƙimar ingancin metagenomic na duk samfuran an daidaita su zuwa maƙallan kowane samfuri daban, wanda ya haifar da adadin adadin rukuni guda biyu waɗanda aka karanta: Tara Marine Viruses - Inriched (190×190), Prokaryotes Enriched (180×180), BioGEOTRACES, HOT da BATS (610×610) da Malaspina (58×58).An yi taswirar ta amfani da Burrows-Wheeler-Aligner (BWA) (v.0.7.17-r1188)54 wanda ke ba da damar karantawa don daidaitawa da rukunin yanar gizo na sakandare (ta amfani da -a tuta).An tace jeri don zama aƙalla tushe 45 tsayi, suna da ≥97% ainihi, kuma tsawon ≥80% karantawa.Sakamakon fayilolin BAM an sarrafa su ta amfani da rubutun jgi_summarize_bam_contig_depths don MetaBAT2 (v.2.12.1)55 don samar da ɗaukar hoto na ciki da tsaka-tsaki ga kowane rukuni.A ƙarshe, an haɗa ɓangarorin don ƙara hankali ta hanyar gudanar da MetaBAT2 daban-daban akan duk samfuran tare da -minContig 2000 da -maxEdges 500. Muna amfani da MetaBAT2 maimakon ɗan dambe mai tarin yawa saboda an nuna shi a cikin gwaje-gwaje masu zaman kansu don zama ɗan dambe ɗaya mafi inganci.kuma sau 10 zuwa 50 cikin sauri fiye da sauran 'yan damben da aka saba amfani da su57.Don gwada tasirin haɗin kai mai yawa, ƙaramin samfurin metagenomics da aka zaɓa bazuwar (10 don kowane ɗayan bayanan Tara Ocean guda biyu, 10 don BioGEOTRAceS, 5 don kowane jerin lokaci, da 5 don Malaspina) kuma an yi amfani da samfuran kawai.Ana haɗa samfuran ciki don samun bayanin ɗaukar hoto.(Ƙarin Bayani).
An haɗa ƙarin kwayoyin halitta (na waje) a cikin bincike na gaba, wato MAGs 830 da aka zaɓa da hannu daga wani yanki na Tara Oceans26 dataset, 5287 SAGs daga GORG20 dataset, da bayanai daga MAR database (MarDB v. 4) daga 1707 ware REFs da 682 SAGs) 27. Don tsarin bayanan MarDB, ana zaɓar nau'ikan kwayoyin halitta bisa la'akari da abubuwan da ake samu idan nau'in samfurin ya dace da maganganun yau da kullun: '[S|s] guda.?[C|c] ell|[C|c] al'ada| [I|i] ware'.
An kimanta ingancin kowane akwati na metagenomic da kwayoyin halitta na waje ta amfani da CheckM (v.1.0.13) da Anvi'o's Lineage Workflow (v.5.5.0) 58,59.Idan CheckM ko Anvi'o ya ba da rahoton ≥50% cikakke / cikawa da ≤10% gurɓatawa/rauni, sannan adana ƙwayoyin metagenomic da ƙwayoyin halittar waje don bincike na gaba.An haɗa waɗannan maki zuwa ma'ana cikakke (mcpl) da ma'anar gurɓatawa (mctn) don rarraba ingancin genome bisa ga ka'idodin al'umma60 kamar haka: babban inganci: mcpl ≥ 90% da mctn ≤ 5%;mai kyau quality: mcpl ≥ 70%, mctn ≤ 10%, matsakaici quality: mcpl ≥ 50% da mctn ≤ 10%, m quality: mcpl ≤ 90% ko mctn ≥ 10%.Sannan an haɗa genome ɗin da aka tace tare da ƙididdiga masu inganci (Q da Q') kamar haka: Q = mcpl – 5 x mctn Q' = mcpl – 5 x mctn + mctn x (saɓanin yanayi)/100 + 0.5 x log[N50].(an aiwatar a cikin dRep61).
Don ba da damar nazarin kwatancen tsakanin tushen bayanai daban-daban da nau'ikan kwayoyin halitta (MAG, SAG da REF), 34,799 genomes an cire su bisa ga matsakaicin matsakaicin ainihin nucleotide (ANI) ta amfani da dRep (v.2.5.4).Maimaitawa) 61 tare da 95% ANI ƙofofin 28,62 (-comp 0 -con 1000 -sa 0.95 -nc 0.2) da kuma kwafin kwafi guda ɗaya ta amfani da SpecI63 yana ba da tari na genome a matakin nau'in.An zaɓi genome mai wakiltar kowane gungu na dRep bisa ga madaidaicin ƙimar inganci (Q') da aka ayyana a sama, wanda aka ɗauke shi wakilcin nau'in.
Don kimanta saurin taswira, an yi amfani da BWA (v.0.7.17-r1188, -a) don taswirar duk saiti 1038 na karatun metagenomic tare da genomes 34,799 da ke cikin OMD.An tsara taswirar karantawa mai inganci a yanayin ƙarewa ɗaya kuma an tace sakamakon da aka samu don riƙe jeri ≥45 bp kawai.da kuma ainihi ≥95%.Matsakaicin nuni ga kowane samfuri shine yawan adadin karatun da suka rage bayan tacewa wanda aka raba da jimillar adadin karatun ingancin inganci.Yin amfani da wannan hanyar, kowane nau'i na 1038 metagenomes an rage shi zuwa 5 miliyan abubuwan da aka saka (bayanan da aka fadada, Fig. 1c) kuma ya dace da GORG SAG a cikin OMD kuma a duk GEM16.Adadin MAGs da aka kwato daga ruwan teku a cikin kasidar GEM16 an ƙaddara ta ta hanyar tambayoyin mahimmin kalmomi na tushen metagenomic, zaɓi samfuran ruwan teku (misali, sabanin ruwan teku).Musamman, mun zaɓi "ruwa" a matsayin "ecosystem_category", "marine" a matsayin "ecosystem_type", da kuma tace "mazauni" a matsayin "teku mai zurfi", "marine", "maritime oceanic", "pelagic marine", "ruwa ruwa" , "Ocean", "Ruwan Teku", "Ruwan Tekun Ruwa", "Ruwan Tekun Ruwa".Wannan ya haifar da 5903 MAGs (mai inganci 734) da aka rarraba akan 1823 OTU (ra'ayoyi a nan).
Prokaryotic genomes an taxonomically annotated ta amfani da GTDB-Tk (v.1.0.2)64 tare da tsoho sigogi niyya GTDB r89 sigar 13. Anvi'o An yi amfani da su gano eukaryotic genomes dangane da yanki Hasashen da kuma tuna ≥50% da redundancy ≤ 10%.An ayyana bayanin taxonomic na jinsi a matsayin ɗaya daga cikin kwayoyin halittarsa.Ban da eukaryotes (148 MAG), kowane nau'in kwayar halitta an fara bayanin shi da aiki ta hanyar amfani da prokka (v.1.14.5)65, suna ba da cikakken suna, yana ma'anar ma'anar "archaea" ko "kwayoyin cuta" kamar yadda ake buƙata, wanda kuma aka ba da rahoton ga waɗanda ba. coding kwayoyin halitta.da CRISPR yankuna, a tsakanin sauran siffofi na genomic.Annotate annabta kwayoyin halitta ta hanyar gano kwayoyin halitta masu alamar kwafi guda ɗaya (uscMG) ta amfani da fetchMG (v.1.2)66, sanya ƙungiyoyin ortholog da tambaya ta amfani da emapper (v.2.0.1) 67 dangane da eggNOG (v.5.0)68.KEGG Database (an buga Fabrairu 10, 2020) 69. An yi matakin ƙarshe ta hanyar daidaita sunadaran sunadaran zuwa bayanan KEGG ta amfani da DIAMOND (v.0.9.30)70 tare da tambaya da ɗaukar hoto na ≥70%.An ƙara tace sakamakon bisa ga NCBI Prokaryotic Genome Annotation Pipeline71 dangane da bitrate ≥ 50% na matsakaicin matsakaicin bitrate (haɗin kanta).An kuma yi amfani da jerin abubuwan halitta azaman shigarwa don gano BGCs a cikin kwayoyin halitta ta hanyar amfani da antiSMASH (v.5.1.0)72 tare da sigogi na asali da fashewar gungu daban-daban.Dukkanin kwayoyin halitta da bayanai an tattara su cikin OMD tare da metadata na mahallin da ake samu akan gidan yanar gizo (https://microbiomics.io/ocean/).
Hakazalika da hanyoyin da aka bayyana a baya12,22 mun yi amfani da CD-HIT (v.4.8.1) don tari> 56.6 miliyan sunadaran kwayoyin-coding daga kwayoyin cuta da kwayoyin halitta daga OMD zuwa 95% ainihi da kuma guntun kwayoyin halitta (90% ɗaukar hoto) 73 har zuwa > Tarin kwayoyin halitta miliyan 17.7.An zaɓi jerin mafi tsayi a matsayin wakilcin halittar kowane tari.Metagenomes na 1038 sannan an daidaita su zuwa> 17.7 miliyan BWA (-a) membobin tari kuma an tace fayilolin BAM da aka samu don riƙe jeri kawai tare da ≥95% kashi na ainihi da ≥45 daidaitattun tushe.An ƙididdige yawan adadin halittu masu tsayi-na al'ada ta farkon kirga abubuwan da aka saka daga mafi kyawun jeri na musamman sannan, don abubuwan da aka saka taswira mai ban mamaki, ƙara ƙididdige juzu'i zuwa madaidaitan kwayoyin halittar da aka yi niyya daidai da adadin abubuwan sakawa na musamman.
Genomes daga OMD da aka faɗaɗa (tare da ƙarin MAGs daga "Ca. Eudormicrobiaceae", duba ƙasa) an ƙara su zuwa mOTUs74 metagenomic analysis Tool database (v.2.5.1) don ƙirƙirar ƙarin bayanan bayanan mOTU.Kwafi guda shida ne kacal (genomes 23,528) suka tsira daga cikin uscMG goma.Fadada ma'ajin bayanai ya haifar da ƙarin gungu 4,494 a matakin nau'in.1038 metagenomes an bincika ta amfani da tsoho mOTU sigogi (v.2).Jimillar kwayoyin halitta 989 da ke kunshe a cikin gungu 644 mOTU (95% REF, 5% SAG da 99.9% na MarDB) ba a gano su ta bayanin martabar mOTU ba.Wannan yana nuna ƙarin maɓuɓɓuka daban-daban na keɓewar ruwa na kwayoyin halittar MarDB (mafi yawan kwayoyin halittar da ba a gano su ba suna da alaƙa da kwayoyin da ke ware daga sediments, rundunonin ruwa, da sauransu).Don ci gaba da mai da hankali kan yanayin buɗaɗɗen teku a cikin wannan binciken, mun cire su daga binciken ƙasa sai dai idan an gano su ko kuma an haɗa su a cikin ƙarin bayanan mOTU da aka ƙirƙira a cikin wannan binciken.
Duk BGCs daga MAG, SAG da REF a cikin OMD (duba sama) an haɗa su tare da BGCs da aka gano a cikin duk ɓangarorin metagenomic (antiSMASH v.5.0, sigogin tsoho) kuma an kwatanta su ta amfani da BiG-SLICE (v.1.1) (PFAM domain)75.Dangane da waɗannan fasalulluka, mun ƙididdige duk nisan cosine tsakanin BGCs kuma mun haɗa su (ma'anar ma'amala) zuwa GCF da GCC ta amfani da matakan nesa na 0.2 da 0.8 bi da bi.Waɗannan ƙofofin daidaitawa ne na ƙofofin da aka yi amfani da su a baya ta hanyar amfani da Euclidean distance75 tare da nisa na cosine, wanda ke rage wasu kuskure a cikin ainihin dabarun tari na BiG-SLICE (Ƙarin Bayani).
Daga nan aka tace BGCs don riƙe ≥5 kb kawai da aka lulluɓe akan tarkace don rage haɗarin rarrabuwa kamar yadda aka bayyana a baya16 kuma don ware MarDB REFs da SAGs waɗanda ba a samo su a cikin metagenomes na 1038 (duba sama).Wannan ya haifar da jimillar BGC 39,055 da OMD genome ke sanyawa, tare da ƙarin 14,106 da aka gano akan gutsuttsuran metagenomic (watau ba a haɗa su cikin MAGs ba).An yi amfani da waɗannan “metagenomic” BGCs don ƙididdige adadin yuwuwar microbiome biosynthesis na ruwa da ba a kama su a cikin bayanan ba (Ƙarin Bayani).Kowane BGC an siffanta shi da aiki bisa ga nau'ikan samfurin tsinkaya da aka ayyana ta anti-SMASH ko nau'ikan samfur mai ƙarfi da aka ayyana a cikin BiG-SCAPE76.Don hana samfurin son zuciya a cikin ƙididdigewa (taxonomic da aikin aikin GCC/GCF, nesa na GCF da GCC don yin la'akari da bayanan bayanai, da yawan adadin GCF), ta hanyar kiyaye BGC mafi tsayi a kowace GCF ga kowane nau'in, 39,055 BGCs an ƙara ƙaddamar da su. wanda ya haifar da jimillar 17,689 BGC.
An tantance sabon sabon abu na GCC da GCF bisa tazara tsakanin bayanan da aka ƙididdige (RefSeq database a cikin BiG-FAM)29 da gwajin gwaji (MIBIG 2.0)30 BGC.Ga kowane wakilin BGCs 17,689, mun zaɓi mafi ƙanƙantar nisan cosine zuwa madaidaitan bayanai.Waɗannan mafi ƙarancin nisa ana ƙididdige su (ma'ana) bisa ga GCF ko GCC, gwargwadon yadda ya dace.Ana ɗaukar GCF sabuwa idan nisa zuwa bayanan bayanai ya fi 0.2, wanda ya yi daidai da kyakkyawar rabuwa tsakanin (matsakaicin) GCF da maƙasudin.Don GCC, mun zaɓi 0.4, wanda shine sau biyu madaidaicin da GCF ya ayyana, don kulle cikin dogon lokaci tare da hanyoyin haɗin gwiwa.
An kiyasata yawan metagenomic na BGC a matsayin matsakaicin yawan adadin kwayoyin halittar halittarsa ​​(kamar yadda anti-SMASH ya ƙayyade) da ake samu daga bayanan martaba-jinni.An ƙididdige yawan yawan metagenomic na kowane GCF ko GCC a matsayin jimlar BGCs (daga 17,689).Waɗannan taswirorin da yawa an daidaita su daga baya don tsarin salula ta amfani da ƙididdige kowane samfurin mOTU, wanda kuma ya yi yunƙurin aiwatarwa (faɗaɗɗen bayanai, siffa 1d).An ƙididdige yawan GCF ko GCC azaman adadin samfuran da ke da yawa> 0.
An ƙididdige nisan Euclidean tsakanin samfuran daga bayanin martaba na GCF da aka saba.An rage waɗannan nisa cikin girman ta amfani da UMAP77 kuma an yi amfani da abubuwan da aka haɗa da su don tari na tushen yawa mara kulawa ta amfani da HDBSCAN78.Mafi kyawun mafi ƙarancin adadin maki don gungu (saboda haka adadin gungu) da HDBSCAN ke amfani da shi ana ƙididdige shi ta hanyar haɓaka yuwuwar haɗaɗɗiyar ƙungiyar tari.Ƙungiyoyin da aka gano (da kuma daidaitaccen misali na waɗannan gungu don yin lissafin son zuciya a cikin nazarin bambance-bambancen da yawa na bambance-bambancen (PERMANOVA)) an gwada su don mahimmanci akan nisan Euclidean mara raguwa ta amfani da PERMANOVA.An ƙididdige matsakaicin matsakaicin girman genome na samfuran bisa la'akari da yawan dangi na mOTU da ƙididdige girman genome na membobin kwayoyin halitta.Musamman ma, an kiyasta matsakaicin girman genome na kowane mOTU a matsayin matsakaicin matsakaicin girman kwayoyin halittar membobinta da aka gyara don cikawa (bayan tacewa) (misali, 75% cikakken genome mai tsayin 3 Mb yana da daidaitaccen girman 4. Mb).ga matsakaici genomes tare da mutunci ≥70%.An ƙididdige matsakaicin matsakaicin girman genome na kowane samfuri azaman jimillar ma'auni na mOTU genome wanda aka auna ta hanyar yalwar dangi.
Tace saitin BGCs na genome-encoded a cikin OMD ana nuna su a cikin bishiyoyin GTDB na kwayan cuta da na archaeal (a cikin tsarin ≥5 kb, ban da REF da SAG MarDB ba a cikin metagenomes na 1038, duba sama) da nau'ikan samfuran da aka annabta bisa tushen phylogenetic. matsayi na kwayoyin halitta (duba sama).Mun fara rage bayanai ta nau'ikan, ta yin amfani da kwayoyin halitta tare da mafi yawan BGCs a cikin wannan nau'in a matsayin wakilci.Don gani, an ƙara rarraba wakilan zuwa ƙungiyoyin bishiya, kuma, ga kowane nau'in tantanin halitta, an zaɓi genome mai ɗauke da mafi yawan adadin BGC a matsayin wakili.Abubuwan da aka wadatar da BGC (aƙalla kwayar halitta guda ɗaya tare da> 15 BGCs) an ƙara yin nazari ta hanyar ƙididdige Ma'anar Diversity Index na Shannon don nau'ikan samfuran da aka sanya a cikin waɗancan BGCs.Idan duk nau'ikan samfuran da aka annabta iri ɗaya ne, samfuran sinadarai da sauran hadaddun BGCs (kamar yadda anti-SMAH ta annabta) ana ɗaukar su na cikin nau'in samfurin iri ɗaya ne, ba tare da la'akari da tsarinsu a cikin gungu ba (misali furotin-bacteriocin da fusion na bacteriocin-proteoprotein). jiki).hybrid).
DNA ɗin da ya rage (wanda aka kiyasta ya zama 6 ng) daga samfurin Malaspina MP1648, daidai da samfurin nazarin halittu SAMN05421555 kuma ya dace da Illumina SRR3962772 metagenomic karanta saitin don taƙaitaccen karantawa, sarrafa shi bisa ga ka'idar tsarin PacBio tare da ultra-low shigarwa don amfani da samfurin GSMNA kit (100-980-000) da SMRTbell Express 2.0 kayan shirya samfuri (100-938-900).A taƙaice, an yanke ragowar DNA, an gyara su kuma an tsarkake su (ProNex beads) ta amfani da Covaris (g-TUBE, 52104).Ana yin DNA mai tsarki don shirye-shiryen ɗakin karatu, haɓakawa, tsarkakewa (ProNex beads) da zaɓin girman (> 6 kb, Blue Pippin) kafin matakin tsarkakewa na ƙarshe (ProNex beads) da jerin abubuwa akan dandamali na Sequel II.
Sake gina na farko biyu ca.Don MAG Eremiobacterota, mun gano ƙarin ANI guda shida> 99% (waɗannan an haɗa su a cikin Hoto na 3), waɗanda aka fara tace su bisa la'akari da ƙima (daga baya an gano su azaman kwafin kwayoyin halitta, duba ƙasa).Mun kuma sami tire mai lakabin "Ca".Eremiobacterota" daga karatu daban-daban23 kuma yayi amfani da su tare da MAGs guda takwas daga bincikenmu azaman ma'anar karatun metagenomic daga samfuran eukaryotic 633 (> 0.8 µm) ta amfani da BWA (v.0.7.17) Ref -r1188, - tuta) don saukarwa. taswira (karanta miliyan 5).Dangane da ƙayyadaddun taswira na wadatarwa (tace ta hanyar 95% daidaitattun daidaito da 80% ɗaukar hoto), an zaɓi metagenomes 10 (ƙirar da ake tsammani ≥5 ×) don haɗuwa da ƙarin 49 metagenomes (ƙirar da ake tsammani ≥1 ×) don daidaita abun ciki.Yin amfani da sigogi iri ɗaya kamar na sama, waɗannan samfuran an ɗaure su kuma an ƙara ƙarin 'Ca's 10.An dawo da MAG Eremiobacterota.Waɗannan MAGs 16 (ba ƙidaya biyun da suka rigaya a cikin bayanan ba) sun kawo jimlar adadin kwayoyin halitta a cikin OMD da aka faɗaɗa zuwa 34,815.MAGs an sanya ma'aunin haraji bisa la'akari da kamanninsu da matsayi a cikin GTDB.18 MAGs an soke su ta amfani da dRep zuwa nau'ikan 5 (ANI na musamman> 99%) da 3 genera (intrageneric ANI 85% zuwa 94%) a cikin dangi guda79.An zaɓi wakilan nau'ikan da hannu bisa aminci, gurɓatawa, da N50.An bayar da shawarwarin ƙididdiga a cikin Ƙarin Bayani.
Tantance mutunci da gurbatar 'Ca.MAG Eremiobacterota, mun ƙididdige kasancewar uscMG, da kuma layi- da ƙayyadaddun ƙayyadaddun kwayoyin halittar kwafi guda ɗaya waɗanda CheckM da Anvi'o ke amfani da su.An tabbatar da gano kwafin 2 daga cikin 40 uscMGs ta hanyar gyare-gyaren phylogenetic (duba ƙasa) don kawar da duk wani gurɓataccen gurɓataccen abu (wannan ya dace da 5% bisa ga waɗannan 40 alamomin kwayoyin halitta).Ƙarin binciken na wakilai guda biyar MAGs 'Ca.An tabbatar da ƙarancin ƙazantattun gurɓatattun ƙwayoyin cuta a cikin waɗannan ƙwayoyin halittar da aka sake ginawa don nau'in Eremiobacterota ta amfani da mu'amalar Anvi'o bisa la'akari da yawa da abubuwan haɗin kai (Ƙarin Bayani)59.
Don nazarin phylogenomic, mun zaɓi MAGs wakilai biyar "Ca".Eudormicrobiaceae, duk nau'in "Ca.Kwayoyin halittar Eremiobacterota da membobin sauran phyla (ciki har da UBP13, Armatimonadota, Patescibacteria, Dormibacterota, Chloroflexota, Cyanobacteria, Actinobacteria da Planctomycetota) yana samuwa daga GTDB (r89)13.Dukkan waɗannan kwayoyin halitta an ƙirƙira su kamar yadda aka bayyana a baya don hakar kwafi guda ɗaya da bayanin BGC.An kiyaye kwayoyin halittar GTDB bisa ga daidaitattun abubuwan da ke sama da ka'idojin gurɓatawa.An yi nazarin phylogenetic ta amfani da aikin Anvi'o Phylogenetics59.An gina itacen ta amfani da IQTREE (v.2.0.3) (zaɓuɓɓukan tsoho da -bb 1000)80 akan daidaitawar sunadaran ribosomal na tandem 39 wanda Anvi'o (MUSCLE, v.3.8.1551)81 ya gano.An rage matsayinsa.don rufe aƙalla kashi 50% na genome82 da Planctomycecota an yi amfani da su azaman rukuni bisa tushen GTDB bishiyar.An gina bishiyar guda 40 na uscMG ta amfani da kayan aiki da sigogi iri ɗaya.
Mun yi amfani da Traitar (v.1.1.2) tare da sigogi na asali (phenotype, daga nucleotides)83 don tsinkayar dabi'un ƙananan ƙwayoyin cuta.Mun binciko yuwuwar salon rayuwa na mafarauta bisa ginshiƙi na farko na farauta index84 wanda ya dogara da abun ciki na kwayar codeing protein a cikin kwayoyin halitta.Musamman, muna amfani da DIAMOND don kwatanta sunadaran da ke cikin genome a kan bayanan OrthoMCL (v.4)85 ta amfani da zaɓuɓɓukan -mafi-m -id 25 -tambayi-rufin 70 -masu-rufin 70 - saman 20 DA ƙidaya kwayoyin halittar da suka dace da su. kwayoyin halitta masu alamar ga mafarauta da wadanda ba masu cin abinci ba.Ma'anar ita ce bambanci tsakanin adadin masu farauta da alamun da ba na gani ba.A matsayin ƙarin sarrafawa, mun kuma bincikar kwayar halittar "Ca".Sakamakon Entotheonella TSY118 ya dogara ne akan haɗin gwiwa tare da Ca.Eudoremicrobium (babban girman genome da yuwuwar biosynthetic).Bayan haka, mun gwada yuwuwar hanyoyin haɗin kai tsakanin mafarauta da ƙwayoyin cuta waɗanda ba masu farauta ba da yuwuwar biosynthetic na Ca.Eudormicrobiaceae" kuma ya gano cewa babu fiye da ɗaya (daga kowane nau'in kwayar halitta mai alama, watau predator/predator gene) ya mamaye BGC, yana nuna cewa BGC baya ruɗe siginar tsinkaya.An yi ƙarin bayani game da ƙayyadaddun ƙididdiga ta amfani da TXSSCAN (v.1.0.2) don bincika musamman tsarin ɓoyewa, pili, da flagella86.
Wakilai 'Ca's guda biyar an tsara ta taswira taswirar metatranscriptomes 623 daga ɓangarorin haɓaka haɓakar prokaryotic da eukaryotic na Tekun Tara22,40,87 (ta amfani da BWA, v.0.7.17-r1188, -a tuta).Eudormicrobiaceae genome.An sarrafa fayilolin BAM tare da FeatureCounts (v.2.0.1)88 bayan 80% karanta ɗaukar hoto da 95% tacewa (tare da fasalin zaɓuɓɓukan ƙididdiga -primary -O -fraction -t CDS,tRNA -F GTF -g ID -p) adadin abubuwan da aka saka a kowane jinsin.Taswirorin da aka ƙirƙira an daidaita su don tsayin jinsin halitta da ma'auni mai yawa mOTU (tsawon matsakaicin matsakaicin matsakaicin matsakaicin shigarwa don kwayoyin halitta tare da ƙididdigewa> 0) kuma an canza log-canza zuwa 22.74 don samun ma'anar dangi ta kowane tantanin halitta na kowane matakin jinsin, wanda kuma yayi bayanin sãɓãwar launukansa daga samfurin zuwa samfurin a lokacin sequencing.Irin waɗannan ma'auni suna ba da damar yin nazari na kwatankwacin, rage matsalolin abun da ke ciki yayin amfani da bayanai masu yawa.Samfurori kawai tare da> 5 na 10 mOTU alamomin kwayoyin halitta an yi la'akari da su don ƙarin bincike don ba da damar gano babban yanki na kwayoyin halitta.
Madaidaicin bayanan martaba na 'Ca.E. taraoceanii an yi shi don rage girman girma ta amfani da UMAP kuma an yi amfani da wakilcin da aka samu don tarawa mara kulawa ta amfani da HDBSCAN (duba sama) don tantance matsayin magana.PERMANOVA yana gwada mahimmancin bambance-bambance tsakanin gungu da aka gano a cikin ainihin (ba a rage) sararin nesa ba.An gwada maganganu daban-daban tsakanin waɗannan yanayi a fadin kwayoyin halitta (duba sama) kuma an gano hanyoyin 201 KEGG a cikin ƙungiyoyi masu aiki na 6, wato: BGC, tsarin ɓoyewa da kuma kwayoyin halitta daga TXSSCAN, enzymes deradation (protease da peptidases), da kuma masu lalata da kuma wadanda ba. kwayoyin halitta masu farauta.alamomin tsinkaya.Ga kowane samfurin, mun ƙididdige maganganun da aka daidaita na tsaka-tsaki ga kowane aji (lura cewa an ƙididdige maganganun BGC da kansa a matsayin tsaka-tsakin maganganun kwayoyin halitta na BGC) kuma an gwada su don mahimmanci a fadin jihohi (gwajin Kruskal-Wallis da aka gyara don FDR).
An sayi kwayoyin halitta na roba daga GenScript kuma an siyi abubuwan farko na PCR daga Microsynth.Phusion polymerase daga Thermo Fisher Scientific anyi amfani dashi don haɓaka DNA.NucleoSpin plasmids, NucleoSpin gel da PCR kayan tsarkakewa daga Macherey-Nagel an yi amfani da su don tsarkakewar DNA.An sayi enzymes ƙuntatawa da T4 DNA ligase daga New England Biolabs.Chemicals banda isopropyl-β-d-1-thiogalactopyranoside (IPTG) (Biosynth) da 1,4-dithiothreitol (DTT, AppliChem) an sayi su daga Sigma-Aldrich kuma an yi amfani da su ba tare da ƙarin tsarkakewa ba.An sayi maganin rigakafi chloramphenicol (Cm), spectinomycin dihydrochloride (Sm), ampicillin (Amp), gentamicin (Gt), da carbenicillin (Cbn) daga AppliChem.Bacto Tryptone da Bacto Yeast Extract abubuwan watsa labarai an siyi su daga BD Biosciences.An siyi Trypsin don jeri daga Promega.
An ciro jerin kwayoyin halitta daga anti-SMASH annabta BGC 75.1.E. malaspinii (Ƙarin bayani).
Kwayoyin halittar embA (wuri, MALA_SAMN05422137_METAG-framework_127-gene_5), embM (wuri, MALA_SAMN05422137_METAG-framework_127-gene_4), da embAM (ciki har da coding na yankuna) an tsara su ba tare da daidaitawa ba a cikin pmpUC. E yaushe.An ƙaddamar da kwayar halittar embA cikin rukunin farko na cloning (MCS1) na pACYCduet-1 (CmR) da pCDFDuet-1 (SmR) tare da wuraren tsagewar BamHI da HindIII.EmbM da embMopt genes (codon-ingantaccen) an sanya su cikin MCS1 pCDFDuet-1 (SmR) tare da BamHI da HindIII kuma an sanya su a cikin rukunin cloning na biyu na pCDFDuet-1 (SmR) da pRSFDuet-1 (KanR) (MCS2) tare da NdeI/ChoI.An sanya kaset ɗin embAM zuwa pCDFDuet1(SmR) tare da wuraren tsagewar BamHI da HindIII.Halin halittar orf3/embI (locus, MALA_SAMN05422137_METAG-scaffold_127-gene_3) an gina shi ta hanyar tsawaitawa PCR ta hanyar amfani da madaidaicin EmbI_OE_F_NdeI da EmbI_OE_R_XhoI, wanda aka narkar da shi tare da NdeI/Xhoted cikin pMC-1 (FDA) enzymes (Ƙarin tebur).6).Ƙuntataccen narkewar enzyme da ligation an yi shi bisa ga ka'idar masana'anta (New England Biolabs).