Name: methods for the extraction of endosymbionts from the whitefly bemisia tabaci
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Finansiel støtte til dette studie blev leveret af National Key Research and Development Program (2016YFC1200601) og National Natural Science Foundation of China (31390421).

1. Hvidfuglbearbejdning og kryptisk artidentifikation <ol><li> Vedligeholdelse af whitefly-arter på bomuld Gossypium hirsutum (Malvaceae) (cv. Zhe-Mian 1973) i bur under standardbetingelser på 27 ± 1 ° C, 70 ± 10% fugtighed og 14 timer lys: 10 timer mørkt regime. </li><li> Indsamle en individuel voksen hvidefly og homogenisere i 30 μl lysisbuffer (10 mM Tris, pH 8,4, 50 mM KCl, 0,45% [vægt / vol] Tween-20, 0,2% [vægt / vol] gelatine, 0,45% [vol / Vol. Nonidet P 40, 60 g / ml Proteinase K). <...

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Since the endosymbionts within whiteflies cannot be cultured in vitro, dissection and assembling bacteriocytes is an effective way to obtain enough genetic material of endosymbionts. Before dissection, the species of whitefly and endosymbionts involved should be explicitly confirmed. The whitefly B. tabaci is a species complex with more than 35 morphologically indistinguishable species and different cryptic species may contain different endosymbionts. Portiera is uniformly harbored as an obliga...

Bakterier der danner et intimt symbiotisk forhold med relative værter er udbredt i leddyr 1 . Endosymbionterne har vist sig at påvirke aspekter af værter, såsom ernæringsmetabolismen, reproduktion, respons på miljøbelastninger 2 , 3 , 4 etc. i næsten alle udviklingsstadier 5 . Imidlertid er mekanismen, der ligger til grund for foreningerne, stadig stort set ukendt. Genomics er af prioritet og betydning, når man studerer de potentielle funktioner og roller af bakterier. Nogle grundlæggende oplysninger, dvs. den taksonomiske status, funktionelle gener, metabolismeveje, sekretionssystemer, kan udledes af genometsekvenser, som kaster lys på symbionernes mulige roller i symbiose. Med udviklingen af ​​høj gennemstrømningssekvensering er et stort antal bakteriegener blevet sekventeret wMed forskellige funktioner afsløret 6 . Endosymbionter er af afgørende betydning i hæmipteraner, såsom bladlus 7 , bedbugs 8 , psyllids 9 , brune plantehoppere 10 og cikader 11 . For eksempel har Buchnera i bladlus, som obligatorisk symbiont, vist sig at være involveret i essentiel aminosyrebiosyntese sammen med generne fra bladlusgenomet 12 . Desuden afsløres transkriptionel regulering af Buchnera 13 . I psyllider er Carsonella sekventeret og rangeret det mindste bakteriegenomet nogensinde fundet 14 . Alle disse karakteristika for endosymbioner er baseret og udledes af genometsekvenserne. Da disse endosymbioner ikke kan dyrkes in vitro , er der blevet anvendt flere fremgangsmåder til isolering af passende bakterier til sequencing. I bladlus ekstraheres endosymbioner gennem centrifugering og filtrering og udsættes for yderligere genomisk og transkriptomisk analyse 5 . I brune plantehoppere sekvenseres endosymbioner sammen med hele insektgenomet 10 . Whitefly B. tabaci er et artskompleks, der indeholder mere end 35 morfologisk ubestridelige arter (kryptiske arter), blandt hvilke to invasive arter har invaderet over hele verden og forårsaget en stor skade for landbrugsproduktionen 15 . Af observeret har endosymbioner inden for B. tabaci- arterne vist betydning i udviklingen af ​​skadegørerne 16 . Til dato er otte endosymbioner blevet identificeret i whiteflyen, herunder obligate symbiont, Candidatus Portiera aleyrodidarum og syv sekundære symbionter Hamiltonella , Rickettsia , Arsenophonus , Cardinium , &lt;Em&gt; Wolbachia, Fritschea og Hemipteriphilus defineret 17 , 18 . I modsætning til de tidligere beskrevne hemipteraner er whitefly B. tabaci et ekstremt lille insekt kun 1 mm i længden. De fleste endosymbioner er begrænset til bakteriocytter 19 (specialiserede celler indeholdende symbionter, som yderligere danner bakteriomer i B. tabaci ). Derudover kan disse endosymbioner ikke dyrkes in vitro . Den eneste måde at opnå endosymbionter fra B. tabaci er at dissekere bakterien ud. Der er dog vanskeligheder med dissektionen. For det første forbinder den skrøbelige bakteriom altid med andre væv fra whiteflyen, hvilket er svært at adskille. For det andet begrænser den lille størrelse af whitefly isoleringen af ​​nok bakteriomer. For det tredje slutter endosymbionter i bakteriomet, hvilket gør det ekstremt kompliceret at erhverve en enkelt bakterieart. <p clAss = &quot;jove_content&quot;&gt; Her rapporterer vi en enkel og billig protokol til at isolere whitefly endosymbionter til efterfølgende metagenom-sekventering. Gennem dissektion, oprensning og amplifikation kunne der opnås tilstrækkeligt endosymbiont-DNA, og bakteriearten kunne bekræftes. Den beskrevne protokol kan anvendes på lignende måde i andre leddyr.

<table border="0" cellpadding="0" cellspacing="0" width="775">
	<tbody>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Taq DNA polymerase</td>
			<td style="width:107px;">Takara</td>
			<td style="width:129px;">R001A</td>
			<td style="width:296px;">including rTaq, 10&#215;Buffer and dNTP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Gel DNA extraction kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">28704</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">DNA sample sequencing system</td>
			<td style="width:107px;">ABI</td>
			<td style="width:129px;">ABI-3730XL</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Microtome</td>
			<td style="width:107px;">Leica</td>
			<td style="width:129px;">EM UC7</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Transmission electron microscopy</td>
			<td style="width:107px;">Hitachi</td>
			<td style="width:129px;">H-7650 TEM</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Stereo microscope</td>
			<td style="width:107px;">Zeiss</td>
			<td style="width:129px;">Stemi 2000-C</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">20 &#956;L microloader</td>
			<td style="width:107px;">Eppendorf</td>
			<td style="width:129px;">F2771951</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter holder</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SX0001300</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Filter membrane filter</td>
			<td style="width:107px;">Millipore</td>
			<td style="width:129px;">SMWP001300</td>
			<td>5.0 &#956;m SMWP</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">REPLI-g UltraFast Mini Kit</td>
			<td style="width:107px;">Qiagen</td>
			<td style="width:129px;">150033</td>
			<td>DNA amlification kit</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">NanoDrop</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:129px;">NanoDrop 2000</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;">Qubit Fluorometer</td>
			<td style="width:107px;">Thermo Fisher Scientific</td>
			<td style="width:129px;">Q33216</td>
			<td></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:243px;">Genome Sequencer</td>
			<td style="width:107px;">Illumina</td>
			<td style="width:129px;">Hiseq 2000</td>
			<td></td>
		</tr>
	</tbody>
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methods for the extraction of endosymbionts from the whitefly bemisia tabaci

Bakterie symbionter danner et intimt forhold til deres værter og giver i de fleste tilfælde fordele til værterne. Genomisk information er kritisk for at studere funktioner og evolution af bakterielle symbioner i deres vært. Da de fleste symbionter ikke kan dyrkes in vitro , er metoder til isolering af en tilstrækkelig mængde bakterier til genom-sekventering meget vigtige. I Whitefly Bemisia tabaci er der blevet identificeret en række endosymbioner, og det forudses at være af betydning for udviklingen og reproduktionen af ​​skadedyrene gennem flere tilgange. Imidlertid forbliver den mekanisme, der ligger til grund for foreningerne, stort set ukendt. Hindringen kommer delvis fra den kendsgerning, at endosymbionterne i whitefly, der hovedsagelig er fastholdt i bakteriocytter, er svære at adskille fra værtscellerne. Her rapporteres en trin-for-trin protokol til identifikation, ekstraktion og oprensning af endosymbionter fra whitefly B. tabaci hovedsagelig ved dissectiPå og filtrering. Endosymbiont-prøver fremstillet ved denne metode, selv om de stadig er en blanding af forskellige endosymbiont-arter, er egnede til efterfølgende genomsekventering og analyse af de mulige roller af endosymbioner i B. tabaci . Denne metode kan også bruges til at isolere endosymbioner fra andre insekter.

Mellemøsten Asien Minor 1 (MEAM1) arter af B. tabaci komplekset blev taget som et eksempel her for beskrivelse. Bomuld til opdræt af hvalfluer og flere udviklingsfaser af hvalfluer er vist i Figur 1, herunder en bomuldsplante, hvide hvidefugle og 1 , 2 og 4 instar nymfer af whitefly (den tredje instar nymf ligner på samme måde som den 4. instar nymf ). Det var indlysende, at den 4. instar nymfe er større end 1 og 2.</...

Watch this Scientific Journal Video about Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci at JoVE.com

Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

Her præsenterer vi en protokol til isolering af endosymbionter fra whitefly Bemisia tabaci gennem dissektion og filtrering. Efter amplifikation er DNA-prøverne egnede til efterfølgende sekventering og undersøgelse af gensidigheden mellem endosymbioner og whitefly.

Metoder til ekstraktion af endosymbionter fra Whitefly<em&gt; Bemisia tabaci</em

Bacterial symbionts form an intimate relationship with their hosts and confer advantages to the hosts in most cases. Genomic information is critical to ...

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Methods for the Extraction of Endosymbionts from the Whitefly Bemisia tabaci

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