Name: detached leaf assays to simplify gene expression studies in potato during infestation by chewing insect manduca sexta
Uploaded: 2019-05-15T12:30:00
Description: Watch this Scientific Journal Video about Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta at JoVE.com

作者要感谢鲍勃·法拉尔和亚历克西斯·帕克提供了在这项研究中使用的昆虫,以及他们在幼虫分期方面的专长。此外,感谢迈克尔·布莱克本和赛卡特·戈什对手稿的批判性评论。
本出版物中提及商品名称或商业产品仅用于提供特定信息,并不意味着美国农业部的建议或认可。
美国农业部是平等机会提供者和雇主。

注: 以下协议是为一个人设置、进行观察和收集样本而设计的。可以组合相同设置的多个运行,以增加生物复制。实验的任何额外重复都应在一天的同一时间进行,以消除对基因表达的可能日影响。该协议旨在为 5 个单独的收获时间点创建 3 个"受感染"的叶子。每个时间点的匹配控制叶总共创建 30 个样本。实验可以进行各种叶片大小和幼虫阶段,但建议叶的大小,幼虫阶段和侵扰时间在整个过程中保持?...

<ol>
	<li>Choi, H. W., Klessig, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DAMPs,+MAMPs,+and+NAMPS+in+plant+innate+immunity.">DAMPs, MAMPs, and NAMPS in plant innate immunity.</a> BMC Plant Biology. 16, 1-10 (2016).</li><li>Pearce, G., Strydom, D., Johnson, S., Ryan, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+polypeptide+from+tomato+leaves+induces+wound-inducible+proteinase+inhibitor+proteins.">A polypeptide from tomato leaves induces wound-inducible proteinase inhibitor proteins.</a> Science. 253, 895-897 (1991).</li><li>Savatin, D. V., Gramegna, G., Modesti, V., Cervone, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wounding+in+the+plant+tissue:+the+defense+of+a+dangerous+passage.">Wounding in the plant tissue: the defense of a dangerous passage.</a> Frontiers in Plant Science. 470 (5), 1-11 (2014).</li><li>Basu, S., Varsanit, S., Louis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altering+Plant+Defenses:+Herbivore-Associated+Molecular+Patterns+and+Effector+Arsenal+of+Chewing+Herbivores.">Altering Plant Defenses: Herbivore-Associated Molecular Patterns and Effector Arsenal of Chewing Herbivores.</a> Molecular Plant-Microbe Interactions. 31, 13-21 (2018).</li><li>Chung, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivore+exploits+orally+secreted+bacteria+to+suppress+plant+defenses.">Herbivore exploits orally secreted bacteria to suppress plant defenses.</a> Proceedings of the National Academy of Sciences, USA. 110, 15728-15733 (2013).</li><li>Chen, M. -. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+direct+plant+defense+against+insect+herbivores:+A+review.">Inducible direct plant defense against insect herbivores: A review.</a> Insect Science. 15, 101-114 (2008).</li><li>Howe, G. A., Major, I. T., Koo, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modularity+in+jasmonate+signaling+for+multistress+resilience.">Modularity in jasmonate signaling for multistress resilience.</a> Annual Review of Plant Biology. 69, 387-415 (2018).</li><li>War, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+defence+against+herbivory+and+insect+adaptations.">Plant defence against herbivory and insect adaptations.</a> AoB PLANTS. 10 (4), 1-19 (2018).</li><li>McCloud, E. S., Baldwin, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivory+and+caterpillar+regurgitants+amplify+the+wound-induced+increases+in+jasmonic+acid+but+not+nicotine+in+Nicotiana+sylvestris.">Herbivory and caterpillar regurgitants amplify the wound-induced increases in jasmonic acid but not nicotine in Nicotiana sylvestris.</a> Planta. 203, 430-435 (1997).</li><li>Schittko, U., Hermsmeier, D., Baldwin, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+between+the+specialist+herbivore+Manduca+sexta+(Lepidoptera,+Sphingidae)+and+its+natural+host+Nicotiana+attenuate:+II.+Accumulation+of+plant+mRNAs+responding+to+insect-derived+cues.">Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuate: II. Accumulation of plant mRNAs responding to insect-derived cues.</a> Plant Physiology. , 701-710 (2001).</li><li>Halitschke, R., Schittko, U., Pohnert, G., Boland, W., Baldwin, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+between+the+specialist+herbivore+Manduca+sexta+(Lepidoptera,+Sphingidae)+and+its+natural+host+Nicotiana+attenuate.+III.+Fatty+acid-amino+acid+conjugates+in+herbivore+oral+secretions+are+necessary+and+sufficient+for+herbivore-specific+plant+responses.">Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural host Nicotiana attenuate. III. Fatty acid-amino acid conjugates in herbivore oral secretions are necessary and sufficient for herbivore-specific plant responses.</a> Plant Physiology. 125, 711-717 (2001).</li><li>Lortzing, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptomic+responses+of+Solanum+dulcamara+to+natural+and+simulated+herbivory.">Transcriptomic responses of Solanum dulcamara to natural and simulated herbivory.</a> Molecular Ecology Resources. 17, 1-16 (2017).</li><li>Hj&#228;lt&#233;n, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simulating+herbivory:+problems+and+possibilities.">Simulating herbivory: problems and possibilities.</a> Ecological Studies. 173, 243-255 (2004).</li><li>Lehtil&#228;, K., Boalt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+and+usefulness+of+artificial+herbivory+in+plant-herbivore+studies.">The use and usefulness of artificial herbivory in plant-herbivore studies.</a> Ecological Studies. 173, 257-275 (2004).</li><li>Schittko, U., Preston, C. A., Baldwin, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eating+the+evidence?+Manduca+sexta+larvae+can+not+disrupt+specific+jasmonate+induction+in+Nicotiana+attenuata+by+rapid+consumption.">Eating the evidence? Manduca sexta larvae can not disrupt specific jasmonate induction in Nicotiana attenuata by rapid consumption.</a> Planta. 210, 343-346 (2000).</li><li>Zebelo, S. A., Maffei, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+early+signalling+events+in+plant-insect+interactions.">Role of early signalling events in plant-insect interactions.</a> Journal of Experimental Botany. 66, 435-448 (2015).</li><li>Maffei, M. E., Mithofer, A., Boland, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Before+gene+expression:+early+events+in+plant-insect+interaction.">Before gene expression: early events in plant-insect interaction.</a> Trends in Plant Science. 12, 310-316 (2007).</li><li>Goodwin, P. B., Adisarwanto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+potato+by+shoot+tip+culture+in+Petri+dishes.">Propagation of potato by shoot tip culture in Petri dishes.</a> Potato Research. 23, 445-448 (1980).</li><li>Goodwin, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+propagation+of+potato+by+single+node+cuttings.">Rapid propagation of potato by single node cuttings.</a> Field Crops Research. 4, 165-173 (1981).</li><li>Martin, P. A. W., Blackburn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+combinatorics+to+screen+Bacillus+thuringiensis+isolates+for+toxicity+against+Manduca+sexta+and+Plutella+xylostella.">Using combinatorics to screen Bacillus thuringiensis isolates for toxicity against Manduca sexta and Plutella xylostella.</a> Biological Control. 42, 226-232 (2007).</li><li>Bell, R. A., Joachim, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Techniques+for+rearing+laboratory+colonies+of+tobacco+hornworms+and+pink+bollworms.">Techniques for rearing laboratory colonies of tobacco hornworms and pink bollworms.</a> Annals of the Entomological Society of America. 69 (2), 365-373 (1976).</li><li>Lawrence, S. D., Novak, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+remarkable+plethora+of+infestation-responsive+Q-type+C2H2+transcription+factors+in+potato.">The remarkable plethora of infestation-responsive Q-type C2H2 transcription factors in potato.</a> BMC Research Notes. 11, 1-7 (2018).</li><li>Green, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PhenoPhyte:+a+flexible+affordable+method+to+quantify+2D+phenotypes+from+imagery.">PhenoPhyte: a flexible affordable method to quantify 2D phenotypes from imagery.</a> Plant Methods. 8 (45), 1-12 (2012).</li><li>Lawrence, S. D., Novak, N. G., Jones, R. W., Farrar, R. R., Blackburn, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivory+responsive+C2H2+zinc+finger+transcription+factor+protein+StZFP2+from+potato.">Herbivory responsive C2H2 zinc finger transcription factor protein StZFP2 from potato.</a> Plant Physiology and Biochemistry. 80, 226-233 (2014).</li><li>Korth, K. L., Dixon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+chewing+insect-specific+molecular+events+distinct+from+a+general+wound+response+in+leaves.">Evidence for chewing insect-specific molecular events distinct from a general wound response in leaves.</a> Plant Physiology. 115, 1299-1305 (1997).</li><li>Browne, R. A., Cooke, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evaluation+of+an+in+vitro+detached+leaf+assay+for+pre-screening+resistance+to+Fusarium+head+blight+in+wheat.">Development and evaluation of an in vitro detached leaf assay for pre-screening resistance to Fusarium head blight in wheat.</a> European Journal of Plant Pathology. 110, 91-102 (2004).</li><li>Browne, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+components+of+fusarium+head+blight+resistance+in+soft+red+winter+wheat+germ+plasm+using+a+detached+leaf+assay.">Evaluation of components of fusarium head blight resistance in soft red winter wheat germ plasm using a detached leaf assay.</a> Plant Disease. 89, 404-411 (2005).</li><li>Michel, A. P., Rouf Mian, M. A., Davila-Olivas, N. H., Canas, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detached+leaf+and+whole+plant+assays+for+soybean+aphid+resistance:+differential+responses+among+resistance+sources+and+biotypes.">Detached leaf and whole plant assays for soybean aphid resistance: differential responses among resistance sources and biotypes.</a> Journal of Economic Entomology. 103, 949-957 (2010).</li><li>Sharma, H. C., Pampapathy, G., Dhillon, M. K., Ridsdill-Smith, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detached+leaf+assay+to+screen+for+host+plant+resistance+to+Helicoverpa+armigera.">Detached leaf assay to screen for host plant resistance to Helicoverpa armigera.</a> Journal of Economic Entomology. 98, 568-576 (2005).</li><li>Vivianne, G. A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+laboratory+assay+for+Phytophthora+infestans+resistance+in+various+Solanum+species+reflects+the+field+situation.">A laboratory assay for Phytophthora infestans resistance in various Solanum species reflects the field situation.</a> European Journal of Plant Pathology. 105, 241-250 (1999).</li><li>Kamoun, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+encoding+a+protein+elicitor+of+Phytophthora+infestans+is+down-regulated+during+infection+of+potato.">A gene encoding a protein elicitor of Phytophthora infestans is down-regulated during infection of potato.</a> Molecular Plant-Microbe Interactions. 10, 13-20 (1997).</li><li>Nowakowska, M., Nowicki, M., K&#322;osi&#324;ska, U., Maciorowski, R., Kozik, E. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Appraisal+of+artificial+screening+techniques+of+tomato+to+accurately+reflect+field+performance+of+the+Late+Blight+resistance.">Appraisal of artificial screening techniques of tomato to accurately reflect field performance of the Late Blight resistance.</a> Plos One. 9, e109328 (2014).</li><li>Arimura, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herbivory-induced+volatiles+elicit+defence+genes+in+lima+bean+leaves.">Herbivory-induced volatiles elicit defence genes in lima bean leaves.</a> Nature. 406, 512-515 (2000).</li><li>Erb, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatiles+as+inducers+and+suppressors+of+plant+defense+and+immunity-origins,+specificity,+perception+and+signaling.">Volatiles as inducers and suppressors of plant defense and immunity-origins, specificity, perception and signaling.</a> Current Opinion in Plant Biology. 44, 117-121 (2018).</li><li>Hasegawa, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+analysis+of+wounding-induced+root-to-shoot+communication+in+Arabidopsis+thaliana.">Gene expression analysis of wounding-induced root-to-shoot communication in Arabidopsis thaliana.</a> Plant, Cell and Environment. 34, 705-716 (2011).</li><li>Ryan, C. A., Moura, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+wound+signaling+in+plants:+A+new+perception.">Systemic wound signaling in plants: A new perception.</a> Proceedings of the National Academy of Sciences, USA. 99, 6519-6520 (2002).</li><li>Hilleary, R., Gilroy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+signaling+in+response+to+wounding+and+pathogens.">Systemic signaling in response to wounding and pathogens.</a> Current Opinion in Plant Biology. 43, 57-62 (2018).</li><li>. Hornworms Available from: <a target="_blank" href="https://www.carolina.com/hornworm/hornworms/FAM_143880.pr">https://www.carolina.com/hornworm/hornworms/FAM_143880.pr</a> (2018)</li><li>. Products Available from: <a target="_blank" href="https://www.greatlakeshornworm.com/products/">https://www.greatlakeshornworm.com/products/</a> (2018)</li><li>. Raising Manduca sexta Available from: <a target="_blank" href="https://acad.carleton.edu/curricular/Biol/resources/rlink/description2.html">https://acad.carleton.edu/curricular/Biol/resources/rlink/description2.html</a> (2018)</li><li>. Teach life cycles with the tobacco hornworm Available from: <a target="_blank" href="https://www.carolina.com/teacher-resources/Interactive/teach-life-cycles-with-the-tobacco-hornworm/tr30179.tr">https://www.carolina.com/teacher-resources/Interactive/teach-life-cycles-with-the-tobacco-hornworm/tr30179.tr</a> (2018)</li><li>Chung, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+plant+species+determines+symbiotic+bacterial+community+mediating+suppression+of+plant+defenses.">Host plant species determines symbiotic bacterial community mediating suppression of plant defenses.</a> Scientific Reports. 7, 1-13 (2017).</li></ol>

使用现有的整个植物食草方法对于实现这一特定研究的目标(即筛选一组候选基因以对感染的反应)是不必要的。分离叶细化的明显好处是缩短了进行草本测定所需的时间。整个植物与夹笼的笨拙性质被消除,并更快地进行检测,因为植物只有2周,可以用来收获叶子。在喂食过程中,它也需要更小的占地面积和更少的生长室空间;当这些资源有限时,两者都很重要。
当对防御基因表达?...

Herbivory 启动一系列分子事件,在此期间,植物可以识别攻击并对其生存作出适当反应。植物从咀嚼昆虫中接收两个基本线索;一个来自对组织的物理损害,另一个来自昆虫特有的物质。损伤相关的分子模式 (DAMPs) 被释放,以响应由幼虫口腔部分造成的损伤,并触发一个明确的伤口反应,导致激素茉莉花酸的增加和防御基因1的转录。其中最著名的DamPs是系统蛋白,一种多肽,由较大的亲系统蛋白的裂解形成后,叶受伤2,3。茉莉花酸伤口反应进一步由草食相关分子模式(HAMPs)调节,可以从毛毛虫唾液、肠道内容物(胃肠)和粪便(frass)4中得出。昆虫使用这些物质来增强或逃避防御反应5。转录因子然后通过调节下游防御基因6,7,8在防御反应中传递来自激素信号的信息。
实验室设置中使用的一些植物-昆虫相互作用研究是模拟类型的,目的是接近昆虫的自然喂养方法。模拟食草动物通常是通过各种工具对植物组织造成人工损伤来实现的,这些工具模拟昆虫嘴部的特定机制,足以引起DAMPs的释放并触发防御基因的产生。其他昆虫特异性成分,如口服分泌物或回流剂,经常被添加来复制从HAMPs9,10,11的贡献。创建特定尺寸和类型的伤口以及应用精确数量的 HAMPs 是这些类型的研究的一个优势,并可提供更可重复的结果。自然草本动物研究,其中对植物组织的损害是通过应用田间获得或实验室饲养的昆虫来完成的,往往更具挑战性,因为伤口大小和HAMP量受昆虫行为控制,并增加了变化数据。自然与模拟方法及其优缺点在文献第12、13、14中得到了很好的争论。
为了研究早期信号事件,如转录因子,必须在相对短的时间内食用一定比例的叶子,因此幼虫必须立即开始咀嚼并保持食用,直到叶子被冷冻进行分析。M. sexta是多索拉动物植物的贪婪的喂食器,在其许多幼虫阶段,使它理想的在相对短的时间内给予最大的伤害15。这在研究早期信号事件时很方便,因为昆虫接触叶面16、17后,植物反应几乎立即发生。常用的夹笼围堵方法证明笨拙,因为多个笼子需要在整个实验过程中不断调整,以便去除或添加幼虫。叶子也必须足够大和足够强大,以支持多个昆虫同时觅食。这些类型的马铃薯植物需要大量的空间来观察喂养。幼虫经常搬迁到叶子表面的底面,这也使得喂食观察相当困难。使用整个工厂进行这些实验显然很麻烦。
目前的研究使用分离的叶子隔离在培养皿,而不是整个植物,以简化和简化整个植物方法,研究草本植物。本研究对该方案的应用仅限于观察M.性幼虫在草食性损伤后马铃薯叶早期诱发的一组C2H2转录因子。

<table border="0" cellpadding="0" cellspacing="0" style="width:1140px;" width="1139">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">agar substitute</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">G3251</td>
			<td style="width:471px;">product is Gelzan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">containment vessel (6,12 or 24 well dish)</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">08-772-49, 08-772-50, 08-72-51</td>
			<td>many other companies sell these products</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca eggs&#160;</td>
			<td style="width:217px;">Carolina Biological Supply Company</td>
			<td style="width:173px;">143880</td>
			<td>30-50 eggs</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">manduca eggs&#160;</td>
			<td style="width:217px;">Great Lakes Hornworm</td>
			<td style="width:173px;">NA</td>
			<td>50, 100, 250 or 500 eggs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca larvae</td>
			<td style="width:217px;">Carolina Biological Supply Company</td>
			<td style="width:173px;">call for specific larval instar requests</td>
			<td>any instar</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">manduca larvae</td>
			<td style="width:217px;">Great Lakes Hornworm</td>
			<td style="width:173px;">call for specific larval instar requests</td>
			<td>any instar</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">microcentrifuge tubes, 1.7 ml&#160;</td>
			<td style="width:217px;">Thomas Scientific</td>
			<td style="width:173px;">1158R22</td>
			<td>these have been tested in liquid N2 and will not explode</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Murashige &#38; Skoog (MS) Basal Medium w/Vitamins</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">M519</td>
			<td>used to make propagation medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">nutrient agar mix</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">M5825</td>
			<td>product is Murashige &#38; Skoog Basal Medium with vitamins, sucrose, and Gelzan</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">paper filter discs</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">09-805A</td>
			<td>Whatman circles-purchase to fit in petri dish</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">petri dish, 60X15 mm or 100X15 mm</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">FB0875713A or FB0875712</td>
			<td>purchase size appropriate for leaf size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">potato tubers&#160;</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">B size (not organic)</td>
			<td style="width:471px;">suggest Maine Farmer&#8217;s Exchange</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">pots, 10&#34;&#160;</td>
			<td style="width:217px;">Griffin Greenhouse Supplies, Inc.</td>
			<td style="width:173px;">41PT1000CN2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">preservative/biocide</td>
			<td style="width:217px;">Plant Cell Technology</td>
			<td style="width:173px;">NA</td>
			<td>product is PPM (Plant Preservative Mixture)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">seed potatoes for explant source</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">B size (not organic)</td>
			<td style="width:471px;">suggest Maine Farmer&#8217;s Exchange</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">slow release fertilizer (14-14-14 )</td>
			<td style="width:217px;">any</td>
			<td style="width:173px;">NA</td>
			<td>Osmocote is a popular brand name</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">soft touch forceps</td>
			<td style="width:217px;">BioQuip</td>
			<td style="width:173px;">4750</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">soil mix</td>
			<td style="width:217px;">Griffin Greenhouse Supplies, Inc.</td>
			<td style="width:173px;">65-51121</td>
			<td>product is Sunshine LC1 mix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">sterile culture vessel&#160;</td>
			<td style="width:217px;">PhytoTechnology Laboratories</td>
			<td style="width:173px;">C2100</td>
			<td>Magenta-type vessel, PTL-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">sterile culture vessel&#160;</td>
			<td style="width:217px;">Fisher Scientific&#160;</td>
			<td style="width:173px;">ICN2672206</td>
			<td>product is MP Biomedicals Plantcon</td>
		</tr>
	</tbody>
</table>

detached leaf assays to simplify gene expression studies in potato during infestation by chewing insect manduca sexta

昆虫食草动物基因表达研究的多营养性需要大量的生物复制,因此需要更简单、更简化的食草动物方案。咀嚼昆虫的扰动通常在整个植物系统中进行研究。虽然这整个有机体策略很受欢迎,但如果类似的观察结果可以在单个分离的叶子中复制,则没有必要。假设信号转导所需的基本元素存在于叶本身中。在信号转导的早期事件的情况下,细胞只需要从扰动接收信号,并将该信号传输到被测定为基因表达的相邻细胞。
建议的方法只是改变了分遣队的时间。在整个植物实验中,幼虫被限制在一片叶子中,最终从植物分离并进行基因表达的测定。如果切除的顺序颠倒,从整个植物研究的最后一个,到分离研究的第一个,喂养实验被简化。
索兰图管龙和葡萄球,Kennebec通过节点转移在简单的组织培养基中传播,并转移到土壤中,如果需要,进一步生长。叶子从母植物被切除,并转移到培养皿,在那里喂养测定进行与M.sexta的幼虫阶段。对受损的叶组织进行测定,以表达信号转导中相对早期的事件。基因表达分析确定了侵扰性特异性Cys2-His2(C2H2)转录因子,确认在早期反应研究中使用分离叶的成功。该方法比整个植物的侵扰更容易执行,并且使用更少的空间。

叶消耗定义协议的成功。健康、准确分级的幼虫应在放置在叶面后立即开始喂食,并且在整个侵扰时间继续以相当一致的方式进食。在视频1中,顶部的幼虫在放置后立即开始咀嚼,并在喂食时保持一致的速率。如果对感染后的早期基因表达事件进行分瓶,这一点尤其重要。底部的幼虫不消耗任何叶物质,是一次不成功的侵扰。
<p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta at JoVE.com

Detached Leaf Assays to Simplify Gene Expression Studies in Potato During Infestation by Chewing Insect Manduca sexta

该方法通过将曼杜卡性幼虫应用于马铃薯分离叶,创造出天然的草食性植物组织受损。植物组织被测定为六个转录因子同源的表达参与早期反应昆虫食草动物。

分离叶测定,以简化马铃薯中基因表达研究,在咀嚼昆虫曼杜卡性塔感染

The multitrophic nature of gene expression studies of insect herbivory demands large numbers of biological replicates, creating the need for simpler, more ...

该协议使用一种简化的方法测量植物基因表达，以回应昆虫食草。通过使用在培养皿中分离的分离叶，昆虫在相对较短的侵扰期更容易应用和监测，有助于可重现的基因表达数据。该协议可能适用于许多植物/昆虫相互作用。如果考虑来自整个工厂系统的基线观测值。昆虫喂养的不可预知性可能具有挑战性。因此，可靠的适当阶段性幼虫和健康植物的供应提供了最好的成功机会。可视化健康的植物和昆虫应该如何出现和行为有助于实现最佳结果，尤其是对于新到研究领域的研究人员。与我展示这个程序的将是弗朗西斯·佩雷斯，一个分子生物学家在水果和蔬菜实验室的基因改进。为了准备节点繁殖组织培养马铃薯植物，首先获得从种植材料中生长的Kennebec植物，至少有三到四个节点。使用无菌刀片去除切割靠近主茎的树枝的叶子，每片留下大约两毫米的枝组织。每个节点上方和下方切割约两毫米，以去除茎上的节点部分，并在含有节点转移介质的无菌组织培养容器中排列节点切口，分支指向上方。然后将节点切割转移到植物组织培养室，在24摄氏度下生长两到三周，并形成16小时光，8小时黑暗照期。为准备昆虫喂养，获得M.sexta所需的幼虫阶段，并根据幼虫的大小将一个幼虫转移到适当密封容器的每个井中。使用五个坚固的托盘，使用五个坚固的托盘，使用五个坚固的托盘，使用五个大小合适的培养皿，用白纸衬里制作放置模板。使用每个纸盒中纸张上适当大小的 Petri 盘跟踪一组六个圆圈，并标记一组控制 A、B 和 C 的圆圈，以及其他受感染的 A、B 和 C。然后用适当的收获时间标记每个放置模板。接下来，在放置模板中为每个圆标一个1.7毫升的微离心管，以适当识别扰动、植物复制信和收获时间点。当所有管子都贴有标签后，将无菌滤纸盘放入每个模板的一个培养盘中，用无菌水润湿光盘，而不会让每个盘内多余的液体池。然后在每个放置模板中放入每个圆圈中放置一个菜，并在每个放置模板旁边放置三个相同年龄和相对大小的马铃薯植物。要启动侵扰，请使用无菌剪刀从每种植物中去除前两个大小匹配的叶子，并尽快将一片叶子放在控制培养皿中，并在受感染的培养皿中放置一片。当所有叶子都放置后，使用柔软的触摸钳子，将至少一个幼虫尽快转移到每个受感染的盘中，并设置所需出虫时间的定时器。观察喂养，以确保所有幼虫都在吃，必要时更换任何非喂养的幼虫。在侵扰时间结束时从所有叶子上去除所有幼虫，然后启动收获计时器。在每个收获时间点结束时，将每片叶子转移到相应的管中，并立即将管子放入液氮中，在零下80摄氏度储存叶样品之前冷冻，直到RNA分离。健康准确分阶段的幼虫应在放置在叶子表面后立即开始喂养，在整个侵扰期间，喂养应以相当一致的方式继续。这种叶子底部的幼虫没有消耗任何植物材料，是一个不成功的侵扰的例子。这些叶子与两周大的节点繁殖组织培养植物分离，每个幼虫阶段以不同的速率和喂养方式食用。根据这些结果，选择第四个星幼虫，以进一步评估对更成熟的土壤种植马铃薯植物的叶子的损害，这些植物的土壤生长更接近田间种植的马铃薯植物。在这些基因表达研究中，确定了两个新的C2H2锌手指转录因子，这些因子对M.sexta食草动物有强有力的反应，支持使用分离叶进行侵扰性检测。请记住，使用年龄和大小匹配的叶子，并正确地阶段每个实验的幼虫作为均匀性导致更可重复的数据。叶组织可以检测为压力感应活性氧物种和茉莉花酸激素衍生物。整个转录组、蛋白质组或微生物组研究也可以在叶组织上进行。

Research

JoVE Journal

Environment

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

使用吉布森大会和基因枪烟草中瞬时基因表达

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

科研

环境科学

A Technique to Screen American Beech for Resistance to the Beech Scale Insect (Cryptococcus fagisuga Lind.)

A Technique to Screen American Beech for Resistance to the Beech Scale Insect Cryptococcus fagisuga Lind.

一种技术，屏幕美国榉木为抵抗山毛榉介壳虫（<em&gt;隐球菌fagisuga</em&gt;林德。）

Beech bark disease (BBD) results in high levels of initial mortality, leaving behind survivor trees that are greatly weakened and deformed.&#160;The ...

Measuring Rates of Herbicide Metabolism in Dicot Weeds with an Excised Leaf Assay

测量除草剂代谢的双子叶杂草价格与切下的叶片分析

In order to isolate and accurately determine rates of herbicide metabolism in an obligate-outcrossing dicot weed, waterhemp (Amaranthus tuberculatus), we ...

Double-stranded RNA Oral Delivery Methods to Induce RNA Interference in Phloem and Plant-sap-feeding Hemipteran Insects

双链 rna 口服法诱导韧皮部和植物-sap 喂养 Hemipteran 昆虫 rna 干扰

Phloem and plant sap feeding insects invade the integrity of crops and fruits to retrieve nutrients, in the process damaging food crops. Hemipteran ...

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

The Barnacle Balanus improvisus as a Marine Model - Culturing and Gene Expression

藤壶Balanus improvisus作为海洋模型的培养和基因表达

Barnacles are marine crustaceans with a sessile adult and free-swimming, planktonic larvae. The barnacle Balanus (Amphibalanus) improvisus is particularly ...

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

A Precise and Autonomous System for the Detection of Insect Emergence Patterns

一种精确、自主的昆虫产生模式检测系统

Existing systems to measure insect emergence patterns have limitations; they are only partially automated and are limited in the maximum number of ...

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

Agrobacterium tumefaciens and Agrobacterium rhizogenes-Mediated Transformation of Potato and the Promoter Activity of a Suberin Gene by GUS Staining

农杆菌和根瘤菌--马铃薯介导的马铃薯转化与葡萄染色诱导素基因的启动子活性

Agrobacterium sp. is one of the most widely used methods to obtain transgenic plants as it has the ability to transfer and integrate its own T-DNA into ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

在纯落叶支架中使用三种截然不同的方法估算叶面积指数

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...